CST Guide:

CST Guide: 

Pathways & Protocols

CST Guide: 

Pathways & Protocols 

First edition 

www.cellsignal.com

Foreword 

The revolution in recombinant DNA that has swept through biomedical research over the past three 

decades has been hailed as an unalloyed advance for almost every aspect of basic biological research. 

The allure of nucleic acid technology, with its clear logic and mathematical precision, has proven irresistible 

for the young researchers who have entered into this field during this period. But it has had its 

downside as well: At the beginning of this era, aspiring research students studied basic biochemistry 

as an essential part of their training. Now, it is viewed by many as an anachronism, a vestige of early 

and mid-20th century experimentation that has been superseded by the far more powerful experimental 

approaches involving manipulation and analysis of nucleic acids. Why study complex mixtures of 

proteins when entire cellular genomes can be sequenced almost overnight 

“use of procedures like these will 

surely support the still-incipient 

campaign of returning to protein 

biochemistry...” 

Dr. Robert A. Weinberg 

The inconvenient truth is that nucleic acid analyses have reached their limits in terms of their ability 

to shed light on the subcellular processes that underlie cell physiology and thus the phenotypes of cells 

and organisms. We are still confronted with the complexities of signal transduction biochemistry that 

underlie almost all biochemical processes. Progress in understanding protein function has lagged far 

behind the molecular biology of nucleic acids, in no small part because studying proteins is so challenging. 

Consider the complexity of a cell in which almost 20,000 distinct genes are being expressed, each 

of which, on average, may specify five distinct protein species because of various alternative splicing 

and post-translational modifications. And then consider the stupefying complexity of how these proteins 

interact combinatorially to create biological function. 

These notions help explain why many have fled protein biochemistry. As is now apparent, this flight 

has left us with an underdeveloped sense of how the protein machinery actually operates to create 

phenotype. As the pressure ramps up to convert basic biomedical discoveries into useful therapeutics, 

our still-inadequate understanding of protein structure and function becomes increasingly apparent. 

Thus, the failure of many ostensibly useful compounds to enter into the clinic can be traced directly to 

our incomplete understanding of the wiring diagram of the cell and how it can be profitably perturbed. 

The present manual represents one very powerful way to reverse this tide. Many of the techniques 

and reagents described here—often involving monoclonal antibodies—can be wielded with the precision 

that nucleic acid mechanics routinely employ. The use of procedures like these will surely support the 

still-incipient campaign of returning to protein biochemistry so that we can indeed learn how things 

really work inside cells. It will require another generation, but the time has come for us to return to the 

old ways, to puzzle out, one protein at a time, how signals are really processed inside cells to create the 

marvelously functioning apparatus—the eukaryotic cell. 

Sincerely, 


Daniel K. Ludwig Professor 

for Cancer Research at MIT 

6 

7

Preface 

“While science is our business, 

as a private company, we can 

focus on doing things beyond 

making money.” 

Dr. Michael J. Comb, PhD 

As a company of scientists for scientists, all of us here at CST are working hard to help you be successful at 

the bench. Over the years we have built information resources to assist you in your research, including the 

PhosphoSitePlus ® PTM database, signaling pathway diagrams, and now this guide. As the CST website has 

taken over the daily function of finding, ordering, and learning about our products, we are phasing out the 

catalog but still wanted to continue to provide a resource to assist you in your daily research activities. We 

hope you find that this guide fulfills that goal. It represents the combined experience of many scientists both 

within and outside of CST. 

I hope you can appreciate that we are a different sort of company. Our goal is not to source every possible 

antibody product or to sell products that we have little, if any, knowledge as to whether or not they work. 

Instead, we focus on producing products in-house so that we can rigorously validate them to ensure that 

they will work as you expect them to. To do this, we have invested heavily in validation and cutting edge 

antibody technologies in order to bring you products of the highest possible quality. We have a large team 

of PhD scientists who build and validate our products, and I encourage you to reach out to them as another 

valuable resource we provide. 

As a private company, we are not driven by quarterly profits. In fact, our goals extend far beyond making 

money; instead, we want to help our customers advance biomedical research. This gives our employees 

freedom to be creative in their workplace, the ability to give back to the local community, and to embrace 

policies and philosophies that are consistent with environmentally responsible views of the world. We’re 

passionate about our mission and excited about partnering with you on the journey ahead. We believe in 

a shared goal—to understand biology at the cellular and molecular level in order to free us of devastating 

diseases like cancer and Alzheimer’s. I’m personally looking very much forward to the next chapter. I hope 

you’ll join us. 

Warm regards, 

Dr. Michael J. Comb, PhD 

CEO & President 

Cell Signaling Technology 

8 www.cellsignal.com/cstceo 

9

Quick 

Reference 

“We are here to help, providing you 

with the products and reference 

materials you need for every step 

of the experimental process.” 

CST Scientists 

Mitochondria; page 18 

Protein Synthesis; page 192 

Vesicle Trafficking; page 248 

Quick Reference 

I: Research Areas 

01 Gene Expression, Epigenetics, and Nuclear Function 20 

02 Signaling 42 

03 Cell Growth and Death 86 

04 Cell Biology 106 

05 Cellular Metabolism 140 

06 Development and Differentiation 152 

07 Immunology and Inflammation 172 

08 Neuroscience 184 

II: Antibody Applications 

09 Chromatin Immunoprecipitation (ChIP) 194 

10 Flow Cytometry (F) 202 

11 Immunofluorescence (IF) 206 

12 Immunohistochemistry (IHC) 214 

13 Sandwich ELISA 220 

14 Western Blotting (WB) 226 

15 Immunoprecipitation (IP) 234 

16 Control Treatments and Cell Lines 238 

III: Workflow Tools 

17 Exploration 250 

18 Investigation 254 

19 Discovery 258 

20 Identification 262 

21 Profiling 264 

22 Modulation 266 

23 Localization & Classification 269 

24 Screening & Quantification 272 

25 Monitoring 274 

26 Verification 277 

27 Customization 278 

Adhesion; page 280 

IV: Additional Information 

28 About CST 282 

29 CST Nature Conservancy 284 

30 Publications by CST Scientists 290 

31 Index 294 

32 Diagram and Table Keys 302 

33 Ordering and Contact Information 304 

See full Table of Contents on pages 12 & 13. 

10 

11

12 

Table of ContentS 

I: Research Areas 

01 Gene Expression, Epigenetics, and Nuclear Function 20 

Chromatin/Epigenetic Regulation 20 

Commonly Studied Chromatin/Epigenetic Regulation Targets 24 

Protein Acetylation Pathway 26 

Histone Lysine Methylation Pathway 27 

Histone Modifications Table 28 

Examples of Crosstalk Between Post-Translational Modifications 31 

Pathway 

Translational Control 32 

Commonly Studied Translational Control Targets 35 

Translational Control: Overview Pathway 36 

Translational Control: Regulation of elF4E and p70 S6K Pathway 37 

Translational Control: Regulation of elF Pathway 38 

Nuclear Receptors 39 

Commonly Studied Nuclear Receptor Targets 39 

Nuclear Receptors Signaling Pathway 41 

02 Signaling 42 

MAP Kinase Signaling 42 

Commonly Studied MAP Kinase Targets 45 

MAPK/Erk in Growth and Differentiation Pathway 44 

Signaling Pathways Activating p38 MAP Kinase Pathway 47 

SAPK/JNK Signaling Pathway 48 

PI3 Kinase/Akt Signaling 49 

Commonly Studied Akt Targets 51 

PI3 Kinase/Akt Signaling Pathway 53 

mTOR Signaling Pathway 54 

Akt Substrates Pathway and Table 55 

Additional Binding Partners Table 65 

Tyrosine Kinases and Associated Phosphatases 66 

Commonly Studied Tyrosine Kinases Targets 69 

Tyrosine Kinases Kinase-Disease Associations Table 71 

ErbB/HER Signaling Pathway 74 

G Protein-Coupled Receptors 75 

Commonly Studied GPCR Targets 77 

G Protein-Coupled Receptor Signaling: Overview Pathway 78 

G Protein-Coupled Receptor Signaling to MAP Kinase/Erk Pathway 79 

Calcium, cAMP, and Lipid Signaling 80 

Commonly Studied Calcium, cAMP, and Lipid Signaling Targets 83 

Calcium, cAMP, and Lipid Signaling Kinase-Disease Associations 84 

Table 

03 Cell Growth and Death 86 

Apoptosis 86 

Commonly Studied Apoptosis Targets 88 

Regulation of Apoptosis Overview Pathway 90 

Inhibition of Apoptosis Pathway 91 

Death Receptor Signaling Pathway 92 

Mitochondrial Control of Apoptosis Pathway 93 

Autophagy 94 

Commonly Studied Autophagy Targets 96 

Autophagy Signaling Pathway 97 

Cell Cycle, Checkpoint Control, and DNA Damage 98 

Commonly Studied Cell Cycle Targets 100 

Cell Cycle/Checkpoint Control Kinase-Disease Associations 102 

Cell Cycle Control: G1/S Checkpoint Pathway 104 

Cell Cycle Control: G2/M DNA Damage Checkpoint Pathway 105 

04 Cell Biology 106 

Adhesion and Extracellular Matrix 106 

Commonly Studied Adhesion Targets 109 

Adherens Junction Dynamics Pathway 110 

Cytoskeletal Regulation 111 

Commonly Studied Cytoskeletal Regulation Targets 113 

Regulation of Actin Dynamics Pathway 115 

Regulation of Microtubule Dynamics Pathway 116 

Protein Folding and VesIcle Trafficking 117 

Commonly Studied Protein Folding and Vesicle Trafficking Targets 119 

Ubiquitin and Ubiquitin-like Proteins 120 

Commonly Studied Ubiquitin Targets 123 

Ubiquitin/Proteasome Pathway 124 

Ubiquitin Ligase Table 125 

Deubiquitinase Table 135 

05 Cellular Metabolism 140 

Commonly Studied Cellular Metabolism Targets 142 

Insulin Receptor Signaling Pathway 144 

Warburg Effect Pathway 145 

AMPK Signaling Pathway 146 

AMPK Substrates Table 147 

06 Development and Differentiation 152 

Commonly Studied Development and Differentiation Targets 158 

Wnt/β-Catenin Signaling Pathway 160 

Notch Signaling Pathway 161 

Hippo Signaling Pathway 162 

Hedgehog Signaling Pathway 163 

TGF-β Signaling Pathway 164 

ESC Pluripotency Differentiation Pathway 165 

Angiogenesis 166 

Commonly Studied Angiogenesis Targets 170 

Angiogenesis Signaling in Tumor Neovascularization Pathway 171 

07 Immunology and Inflammation 172 

Commonly Studied Immunology and Inflammation Targets 174 

Jak and Cytokine Receptor Mutants Table 176 

Jak/Stat Utilization Table 177 

B Cell Receptor Signaling Pathway 178 

T Cell Receptor Signaling Pathway 179 

Toll-like Receptor Signaling Pathway 180 

Jak/Stat Signaling: IL-6 Receptor Family Pathway 181 

NF-κB Signaling Pathway 182 

Tumor Immunology Pathway 183 

08 Neuroscience 184 

Commonly Studied Neuroscience Targets 187 

Vesicle Trafficking in Presynaptic Neurons: Synchronous Release 189 

Pathway 

Amyloid Plaque and Neurofibrillary Tangle Formation in Alzheimer’s 190 

Disease Pathway 

Dopamine Signaling in Parkinson’s Disease Pathway 191 

II: Antibody Applications 

09 Chromatin Immunoprecipitation (ChIP) 194 

ChIP General Protocol 196 

ChIP Troubleshooting Guide 200 

10 Flow Cytometry (F) 202 

Flow Cytometry General Protocol 204 

Flow Cytometry Alternate Protocol (for combined staining 

204 

of intracellular proteins and cell surface markers in blood) 

11 Immunofluorescence (IF) 206 

Cultured Cells (Immunocytochemistry, IF-IC) Protocol 210 

Frozen/Cryostat Tissue Sections (IF-F) Protocol 211 

Paraffin Tissue Sections (IF-P) Protocol 212 

In-Cell Western Protocol 213 

12 Immunohistochemistry (IHC) 214 

IHC Paraffin Protocol (using SignalStain ® Boost Detection Reagent) 216 

IHC Frozen Protocol (using SignalStain ® Boost Detection Reagent) 217 

IHC Troubleshooting Guide 219 

III: Workflow Tools 

17 Exploration 250 

Using PhosphoSitePlus ® online database 

18 Investigation 254 

Using motif and post-translational modification-specific antibodies 

19 Discovery 258 

Using peptide immunoaffinity enrichment and LC-MS/MS to study 

post-translational modifications (PTMs) 

20 Identification 262 

Using Chromatin IP (ChIP) to identify protein-DNA interactions 

21 Profiling 264 

Using antibody arrays 

22 Modulation 266 

Using chemical activators and inhibitors, cytokines, and growth factors 

IV: Additional Information 

28 About CST 282 

Behind the Antibodies 282 

Corporate Social Responsibility 282 

Sustainability 283 

Environmental Awareness and Support 283 

29 CST Nature Conservancy 284 

Conservancy Efforts & Resources 284 

30 Publications by CST Scientists 290 

31 Index 294 

Pathway Diagrams and Tables 294 

Protocols and Troubleshooting Guides 294 

Targets 295 

Trademarks, Terms and Conditions 301 

13 Sandwich ELISA 220 

PathScan ® Sandwich ELISA Colorimetric Protocol 221 

PathScan ® Sandwich ELISA Chemiluminescent Protocol 223 

PathScan ® Sandwich ELISA Antibody Pair Protocol 224 

14 Western Blotting (WB) 226 

WB General Protocol 228 

WB Fluorescent Protocol 230 

WB Troubleshooting Guide 232 

15 Immunoprecipitation (IP) 234 

IP Native Protein Protocol 235 

IP Denatured Protein Protocol 237 

16 Control Treatments and Cell Lines 238 

23 Localization & Classification 269 

Using conjugated primary antibodies 

24 Screening & Quantification 272 

Using ELISA to screen and quantify cellular responses at the target level 

25 Monitoring 274 

Using cell assays to monitor the health of cells and their response to 

multiple challenges 

26 Verification 277 

Using siRNA-mediated knockdown to verify function 

27 Customization 278 

Antibodies in a carrier-free formulation for specific assay platforms 

32 Diagram and Table Keys 302 

Pathway Diagram Key 302 

Application Key 303 

Reactivity Key 303 

Amino Acid Properties Table 303 

33 Contact Information 304 

Global Headquarters 304 

Ordering and Customer Service 304 

Technical Support 304 

13

ippo 

GF 

1011010100110101101100101 

0 10 1 0101 

0 10 10 10 1 

1011010100110101101010101 

0 10 1 0101 

otch 

Pathway 

Series 

Table of ContentS 

Revised Edition 

Angiogenesis 

Wnt Signaling 

H g 

Signalin 

GPCR Signaling 

R-spondin 

R-spondin 

T β g 

Signalin 

Frizzled 

Wnt1 

Wnt5A 

Frizzled 

LRP5/6 

mut 

exp 

mut 

Receptor Tyrosine Kinase (RTK) 

mut, trans, amp, exp 

ligand 

E-cadherin 

CD44 

FAT4 

BMPRI 

BMPs 

BMPRII 

mut 

GPCR 

GPCR 

GPCR 

α 

Ras 

γ 

β 

γ 

β 

α 

PLCβ 

ZNRF3/RNF43 

CKI 

PAR-1 

CKI 

LGR4,5,6 

p120 

AC 

Src 

β-catenin 

α 

[IP 3 ] 

PLCβ 

α-catenin 

γ 

β 

[Ca 2+ ] 

[DAG] 

PI3Kγ 

PI3K 

Ubiquitin 

Crk 

C3G 

[cAMP] 

Dsh Akt 

Dsh 

PAR-1 

GSK-3β 

SOS 

GRB2 

EPAC 

CAMKII 

PKC 

PP2A 

APC 

WTX 

Axin 

SARA 

Smad2/3 

Smad2/3 

TGFβ RI 

TGFβ 

TGFβ RII 

Smad7 

mut 

mut 

mut 

mut 

GPCR 

mut 

α s 

Ras 

VEGF 

EPO 

PDGF 

mut 

mut 

α 12/13 

γ 

β 

Smad1/5/8 

mut 

mut 

ALK 

ROS1 

EGFR 

HER2 

mut 

mut 

14-3-3 

mut 

FRMD 

Mer 

KIBRA 

ERK 

mut 

Smad7 

mut 

Smad1/5/8 

mut 

Ras Signaling 

amp, mut, exp 

Rap 

[PIP 3 ] 

β-catenin 

14-3-3 

YAP 

Mst1/2 

SAV1 

TAK 

Smad4 

PKA 

Ras 

NF1 

B-Raf 

14-3-3 

Raf-1 

GRB2 

SOS 

Normoxia 

Hypoxia 

SynGAP 

RasGRF 

RasGRP 

RasGAP 

Ubiquitin 

TAB1 

TAB2 

mut 

mut 

mut 

TAB1 

TAB2 

mut 

LATS1/2 

MOB1 

RasGEF 

Integrin 

RasGAP 

HPH 

Ras c-Raf 

Akt 

RAR 

SOX 

β-catenin 

YAP/TAZ 

NLK 

Smad1/5/8 

Smad4 

Smurf 

mut, del 

mut 

Smad2/3 

Smad4 

mut 

mut 

PI3K ILK 

Src 

FAK 

GRAF 

RIAM/Talin 

RhoGEFs 

KSR 

14-3-3 c-Raf 

MEK1/2 

ERK1/2 

MP1 

CBP 

TAK p38 

MKK3/6 

amp, mut, exp 

mut 

amp 

mut 

E2F 

C 

amp, exp 

β-catenin 

TCF/LEF 

MKK7 JNK 

VHL 

RalGEF 

RalB 

HIF-1α(-2α) 

HIF-1β 

other TFs 

TEAD 

YAP/TAZ 

Smad 

mut 

del 

ERK1/2 

Smad1/5/8 

Smad4 

mut 

mut 

mut 

myc, cyclin D, CD44 

exp 

p107 

E2F 

Smad2/3 

Rap 

RalA 

CDK7 

Cyclin H 

RalBP1 

Regulated 

Exocytosis 

Exocyst 

Complex 

Rab8a (c-Mel) 

Cdc42 Actin 

Rac 

PAK1 

Cytoskeleton 

JNK 

MEK1/2 

MP1 

ERK1/2 

p90 RSK 

p90 RSK 

Rho 

ERK1/2 

CREB 

HIF-1α(-2α) 

HIF-1β 

Fos 

Jun 

myc, cyclin D 

Cdc2 

Cyclin B 

p15, p21, p27 

mut 

mut 

exp 

Wee1A 

Miz-1 

Smad2/3 

Smad4 

mut, amp 

HPV-E7 

DNA Repair 

Cdc25 

B/C 

Myc 

Miz-1 

Rad52 

Rad51 

JNK 

NBS1 

Myc 

Max 

autophagosome 

Myc 

Max 

cyclin D, myc 

FANC 

D2 

HDAC 

Rb 

DP E2F 

BRCA1 

cyclin D, myc 

mut, amp 

vir 

mut 

mut, del 

Atg16/12/5 

mut 

SQSTM1 

PI3K 

class III 

Beclin 

Atg14 

exp 

Autophagy 

p90 RSK 

mut, amp 

Fos 

Jun 

phagopore 

membrane 

nucleation 

Elk-1 

CDK4/6 

Cyclin D 

4E-BP 

p15 p16 p18 p19 

mut 

UV 

DNA Damage 

Cdc2 

Cyclin A 

S6K 

SHP1 

GSK-3 

Cyclin D p21 

STAT STAT 

DP E2F 

IR 

* * 

Abl 

ULK1 

FIP200 

Atg13 

mut 

amp, trans 

Chk1 

ATM 

ATR 

Chk2 

Cdc25A 

DNA- 

PK 

MDM2 

Nutrients 

JAK 

JAK 

STAT 

SHP2 

GRB2 

STAT 

SOS 

Shc 

Ras 

(amino acids 

& glucose) 

Rb 

(Inactive) (Active) 

trans 

mut 

LC3-II 

mut 

LC3-I 

mut 

mut mut 

mut,del 

Scienstists at Ce l Signaling Technology co laborate 

with key opinion leaders in cancer research to create 

reference pathway diagrams that reflect the latest 

thinking in the research community. Over 40 pathway 

diagrams currently exist, including the pathways 

represented here and many others. 

www.cellsignal.com/CSTcancer 

mut 

Atg7/3 

Atg4 

mut,del 

p53 

p21 

trans, mut 

mut 

p53 

TSC2 

TSC1 

mTOR 

Raptor 

amp 

Rheb 

AMPK 

p27 

FKHR/ 

FOXO 

cyclin D 

mut 

Cytokine 

Receptor 

mTOR 

Rictor 

Cdc25A 

R 

cyclin E, A 

myc, cdc2 

p107, E2F 

mut 

The Pathways in Human Cancer poster summarizes some of the key 

signaling pathways implicated in tumorigenesis and tumor 

progression in humans. Within each pathway, gene products known 

to be mutated in human tumors—oncogenes and tumor suppressor 

genes—are coded with information on types of genetic alterations 

and conferred capabilities to the tumor. Proteins are shown using 

structural representatives. New to this revised poster edition are 

signaling pathways for Hippo Signaling, Autophagy, Warburg Effect, 

and Epigenetic Regulation. 

This poster was created by scientists at Cell Signaling Technology in 

collaboration with Robert A. Weinberg and others at the forefront of 

cancer research. Expanded versions of each pathway, including 

additional downstream signaling nodes, can be found at 

www.cellsignal.com/CSTcancer. 

HIPK2 

ARF 

MDM2 

mut 

mut 

CDK7 

Cyclin H 

trans 

LKB1 

Aurora 

A 

HPV-E6 

mut 

amp 

amp, exp 

vir 

Transcription 

Akt 

PI3K 

PDK1 

Lamins 

PP2A 

14-3-3 Bim 

XIAP 

Bad 

MDM2 Bcl-x L 

p53 

CDK2 

Cyclin A 

amp, mut, exp 

amp 

Casp-2 

mut,del 

amp,exp 

CDK2 

Cyclin E 

p21 

p27 

mut 

myc, cyclin D, cyclin E 

mut 

mut 

Bax 

Bcl-2 

NOXA 

PUMA 

c-Raf 

Smac 

PARP 

cyclin D 

trans 

CAD 

Gli PKA 

KMT2/MLL 

KDM6A/UTX 

KMT4/DOT1L 

BRD4 

TET 

SirT1 

SWI/SNF 

KMT6/EZH2 

DNMT3 

HDAC 

Sin3 

CSL/CBF1 

CSL/CBF1 

amp, trans 

p300 

Epigenetic Signaling 

AIF 

cIAP 

NFκB1/2 

RelA 

NFκB2 

RelB 

Ce l Signaling Technology would like to thank 

digizyme for their co laboration on the design 

and concept of this poster. Please visit 

www.digizyme.com to see more of their work. 

0 101010110110110 

mut 

NICD 

mut,del 

PKM2 

[PIP 3 ] 

p53 

Apaf-1 

Casp-9 

Casp-3,6,7 

ICAD 

CAD 

IκB 

HAT HES1 

Gab1 

PI3K 

RTK 

Gab2 

IRS-1 

Cbl 

PTEN 

PI3K 

pyruvate 

LDHA 

Akt Signaling 

mut 

lactate 

PFK 

Fructose 

Bisphosphate 

NICD 

amp, trans 

Gli 

mut 

PDK1 

Ras 

Gli 

tBid 

Bcl-2 

Su(Fu) 

KIF-7 

Su(Fu) 

KIF-7 

Bax 

MEKK3 

JNK 

FLIPs 

NUMB 

Dsh 

Presenilin 

ASK 

Bak 

Bid 

TRADD 

CytC 

NFκB1/2 

RelA 

IκB 

[PIP 3 ] 

Hexokinase 

G-6-P 

F-6-P 

IDH1 

mut, del 

amp, trans 

Fatty acid 

synthesis 

Krebs 

Cycle 

TAK 

IKKα IKKβ 

mut 

citrate 

IDH2 

trans 

NEMO 

Ras 

TAB2 

TAB3 

IKKα 

IKKβ 

Glucose 

transporter 

citrate 

mut 

mut 

mut 

MKK7 

NIK 

Direct stimulatory modification 

Direct inhibitory modification 

Multistep stimulatory modification 

Tentative stimulatory modification 

Transcriptional contribution 

TYPES OF GENETIC ALTERATIONS: 

Point mutation 

Amplification 

Translocation 

Deletion 

Viral infection 

Increased expression 

(unknown mechanism) 

mut 

amp 

trans 

del 

vir 

exp 

TYPES OF CONFERRED CAPABILITIES: 

Evading apoptosis 

Self-sufficiency in growth signals 

Insensitivity to anti-growth signals 

Tissue invasion & metastasis 

Limitless replicative potential 

Sustained angiogenesis 

∞ 

Ras Common protein name 

mut 

Structural representative 

from PDB 

oncogene 

tumor suppressor 

Type of genetic alteration 

Type of conferred capability 

Warburg Effect 

Akt 

FADD 

Casp-8,10 

amp, mut, exp 

IRAK 

TRAF6 

Myd88 

RIP TRADD 

RAIDD 

TRAF2 

TRAF 

2/6 

TRAF 

3 

Notch 

Furin 

Fringe 

NIC 

mut 

mut 

mut 

mut 

Fas/DR 

Ptch 

Cleaved 

Notch 

Smo 

Ptch 

Smo 

mut 

IL1R 

TNFR 

TNFR 

CDO 

BOC 

Hh 

trans 

Delta 

Jagged 

D eath Receptor / NF- κB g 

TACE 

trans 

CDO 

BOC 

This poster accompanies the textbook The Biology of Cancer, Second Edition 

by Robert A. Weinberg and published by Garland Science. 

Dr. Weinberg is a founding member of the Whitehead Institute for Biomedical 

Research and the Daniel K. Ludwig Professor of Cancer Research at the 

Massachusetts Institute of Technology. 

Signalin 

S ignalin 

N g 

Hardcover ISBN 978-0-8153-4219-9 

Paperback ISBN 978-0-8153-4220-5 www.garlandscience.com 

To request free copies of this poster please visit our website. www.cellsignal.com/phcposter 

H edgehog g 

Signalin 

Annual 

Signaling 

in Cancer 

Symposium 

Cell Signaling Technology is proud to partner with the 

MIT’s Koch Institute to present this Annual Symposium, 

which brings together cancer research thought leaders 

from around the world to share current findings and 

further research community collaboration. 

Speakers focus on the cancer pathways that support 

tumor development, the emerging research in identifying 

and targeting these pathways, and innovations 

in the development of increasingly effective cancer 

therapy options. 

CST and MIT’s Koch Institute 

Proudly Sponsor the Targeting 

Cancer Pathways Webinar Series 

in collaboration with Science and Science Signaling 

Targeting Cancer Pathways: 

Recent advances in our understanding of cancer 

have revealed that the disease cannot be understood 

through simple analysis of genetic mutations within 

cancerous cells. Instead, tumors should be considered 

complex tissues in which the cancer cells communicate 

with the surrounding cellular microenvironment 

and evolve traits that promote their own survival. 

Signaling pathways mediate the cross-talk between 

tumor-intrinsic factors, such as genomic instability, 

and tumor-extrinsic factors, such as inflammation, 

driving cancers to acquire the hallmark capacities to 

sustain proliferation, downregulate tumor suppressors, 

evade the immune system, resist cell death and 

senescence, induce angiogenesis, reprogram energy 

metabolism, and metastasize to secondary sites. 

This seminar series focuses on the cancer pathways 

that support tumor development, the emerging 

research in identifying and targeting these pathways, 

and innovations in the development of increasingly 

effective cancer therapy options. 

Please visit our website for more information 

and to register for upcoming symposiums. 

www.cellsignal.com/cstkoch 

14 

In collaboration with 

www.cellsignal.com/cstkoch 

15

125 

of Antibody Discovery and Development 

We celebrate the 125th anniversary 

of the first use of antibody-based therapy. 

From early explorations of vaccination to present-day clinical trials, antibody-based research 

and therapies have long demonstrated their enormous potential to benefit human health. 

1890 

Antibodies are 

shown to be active 

against diphtheria 

and tetanus, giving 

rise to a humoral 

theory of immunity 

(Emil von Behring 

and Kitasato 

Shibasaburo) (1) 

1891 

Observe transferrable 

immunity (Emil von 

Behring and Kitasato 

Shibasaburo) (2) 

1896 

Jules Bordet identifies 

complement as an 

antibacterial, heat-labile 

serum component (3) 

1940 

Karl Landsteiner 

and Alexander 

Weiner identify 

Rh antigens (8) 

1938 

John Marrack 

proposes Antigen- 

Antibody binding 

hypothesis (6) 

1942 

Albert Coons labels antibodies with FITC 

originating the field of immunofluorescence (9) 

Jules Freund and Katherine McDermott demonstrate 

use of adjuvants to stimulate antibody production (10) 

1957 

Clonal selection 

theory proposed by 

Frank MacFarlane 

Burnet and David 

W. Talmage (14,15) 

Free Antibody History Poster... 

www.cellsignal.com/abhistory 

1890 1900 1940 1955 1965 1975 1980 1990 1995 

2000 

2010 2015 

1939 

1948 

1971 

Arne Tiselius and Elvin 

Astrid Fagreaus 

1960 

ELISA assay 

1986 

2002 

Kabat discover the 

discovered antibody 

Radioimmunoassay 

developed 

Sean P. O’Neill and Joseph Wu 

CST receives patent for 

first antibody isotype, 

production in 

developed by Rosalyn 

independently by 

awarded patent for quantitative 

motif antibody technology (41) 

gamma-globulin (7) 

plasma B cells (11) 

Yalow and Solomon 

Eva Engvall and 

Berson (18) 

immunoprecipitation assay (33) 

Peter Perlman 

(21) 

1900 

Paul Erlich develops antibody 

formation theory (4) 

1909 

Almroth Wright publishes, 

“Studies on Immunisation”, 

a collection of papers 

describing opsonization in 

the context of therapeutic 

immunization (5) 

1944 

IgM is described 

independently by 

Jan Waldenström with 

Kai Pedersen as well 

as Henry Kunkel (7) 

1955 

Niels Jerne 

proposes 

naturalselection 

theory 

of antibody 

formation (13) 

1965 

Thomas Tomasi identifies secretory 

immunoglobulins (IgA) (19) 

First fluorescence based 

assay developed by 

Martin Fulwyler (20) 

1953 

Wallace Coulter awarded a 

patent on Coulter principle, 

enabling flow cytometry (12) 

1959 –1962 

Antibody structures independently 

elucidated 

by Gerald Edelman and 

Rodney Porter (16,17) 

1956 

Kappa and lambda light 

chains, then known as 

Bence Jones proteins, 

are shown to be two 

separate proteins by 

Leonard Korngold and 

Rose Lipari (7) 

1976 

Susumu Tonegawa describes 

somatic recombination of 

immunoglobulin genes 

to account for incredible 

diversity (24) 

1972 

FACS instrument 

developed and 

patented by Len 

Herzenberg’s 

lab at Stanford 

University (22) 

1966 

Kimishige Ishizaka et al. 

and S.G.O. Johansson & 

Hans Bennich independently 

identified IgE as 

the reaginic 

antibody (7) 

1975 

Georges Köhler and 

César Milstein develop 

hybridomas leading to the 

production of mAbs (23) 

1967 

Kimishige Ishizaka identifies 

IgE as the reaginic antibody, 

binding the molecule that 

induced its synthesis (7) 

1978 

Hybritech becomes the 

first mAb company (25) 

1979 

First patent on hybridoma 

technology awarded to Wistar 

Institute (26) 

Western blotting, perhaps the 

most widely used immunoassay 

in research, is invented by Harry 

Towbin et al. (27) 

1982 

Angus Nairn, et al. 

develop the first 

phospho-specific 

antibodies (30) 

1985 

John Lis and David 

Gilmour develop Chromatin 

Immunoprecipitation (ChIP) 

assay (32) 

1984 

hCG antibodies used to develop 

5 minute pregnancy test (31) 

1995 

Katherine Knight and 

colleagues at Loyola 

University, Chicago, 

USA published first 

paper on rabbit mAb 

development (35) 

1990 

John McCafferty et al. 

report the use of phage 

display for antibody 

discovery (34) 

1996 

Prostascint ® , radiolabeled 

anti-PSMA 

(prostate specific 

membrane antigen) 

imaging antibody 

approved by the 

FDA (36) 

1981 

The lab of Herman Eisen develops the first anti-pTyr antibody (28) 

Hybritech delivers first mAb product to measure IgE in blood to 

diagnose allergic reactions (29) 

1997 

Idec markets the world’s first mAb 

treatment for lymphoma (Rituxan ® ) (37) 

1998 

Herceptin ® approved for 

breast cancer treatment (38) 

2004 

Erbitux approved by 

FDA for treatment of 

colorectal cancer (42) 

2006 

CST releases its first 

antibody developed 

using the proprietary 

XMT method (43) 

2000 

Abgenix develops XenoMouse ® 

which produces fully human 

antibodies (40) 

1999 

CST established as an independent 

company and releases its first kinase 

substrate motif antibody (#9611) (39) 

2014 

Yervoy ® (ipilimumab), a 

monoclonal anti-CTLA4 

antibody and the first 

immune checkpoint 

cancer therapy, receives 

FDA approval as 

a late-stage melanoma 

treatment (46) 

2013 

Kadcyla ® (ado-trastuzumab 

emtansine), an antibodydrug 

conjugate, receives 

FDA approval as late-stage 

breast cancer treatment (45) 

2012 

CST publishes NG-XMT 

method, a proteomics 

approach to developing 

mAbs (44) 

16 

References: 

1. von Behring, E., and Shibasaburo, 

K. (1890) Deutsche Medizinsche 

Wochenschrift 16, 1113–1114. 

2. Deshpande, S.S., Enzyme Immunoassays: 

From Concept to Product 

Development. US: Springer; 1996. 

3. Jules Bordet - Biographical. Nobelprize.org. 

www.nobelprize.org/nobel_prizes/medicine/laureates/1919/ 

bordet-bio.html. 26 Sep 2014. 

4. Abbas, A.K., Lichtman, A.H., Cellular 

and Molecular Immunology. 2005. 

Philadelphia, PA: Elsevier; 2005. 

5. Turk, J.L. (1994) J. R. Soc. Med. 87, 

576–577. 

6. Marrack, J. The Chemistry of 

Antigens and Antibodies. London, 

UK: H.N. Stationery Off; 1938. 

7. Reviewed in Black, C.A. (1997) 

Immunol. Cell Biol. 75, 65–68. 

8. Landsteiner, K. and Wiener, A.S. 

(1940) Proc. Soc. Exp. Biol. Med. 

43, 223–224. 

9. McDevitt, H.O. and Albert Hewett 

Coons 1912–1978: A Biographical 

Memoir. Washington D.C.: National 

Academies Press; 1996. 

10. Reviewed in Bendelac, A. and 

Medzhitov, R. (2002) J. Exp. Med. 

195, 19–23. 

11. Fagreaus, A. (1948) J. Immunol. 

58, 1–13. 

12. US Patent No. 3,557,352 January 

19th, 1971. 

13. Jerne, N.K. (1955) Proc. Natl. 

Acad. Sci. USA 41, 849–857. 

14. Burnet, F.M. (1957) Aust. J. Sci. 

20, 67–69. 

15. Talmage, D.W. (1957) Annu. Rev. 

Med. 8, 239–256. 

16. Edelman, G.M. and Poulik, M.D. 

(1961) J. Exp. Med. 113, 861–884. 

17. Milstein, C. (2007) Int. 

J. Immunogen. 13, 1–2. 

18. Yalow, R.S. and Berson, S.A. (1960) 

J. Clin. Invest. 39, 1157–1175. 

19. Reviewed in Tomasi, T.B. (1992) 

Immunol. Today 13, 416–418. 

20. Fulwyler, M.J. (1965) Science 150, 

910–911. 

21. Engvall, E. and Perlman, P. (1971) 

Immunochem. 8, 871–874. 

22. Reviewed in Hardy, R.R. and 

Roederer, M. (2013) Cell. Immun. 

39, 989–991. 

23. Köhler, G. and Milstein, C. (1975) 

Nature 256, 495–497. 

24. Reviewed in Gearhart, P.J. (2004) 

J. Immunol. 173, 4259. 

25. Chi, K.R. (2007) The Scientist, www. 

the-scientist.com/articles.view/ 

articleNo/25737/title/The-Birth-of- 

Biotech/ 

26. Ansell, P.R.J. (2000) Immunol. 

Today 21, 357–358. 

27. Towbin, H. et al. (1979) Proc. Nat. 

Acad. Sci. USA 76, 4350–4354. 

28. Ross, A.H., Baltimore, D., and 

Eisen, H.N. (1981) Nature 294, 654. 

29. US Patent No. 4,376,110 

30. Nairn, A.C. Detre, J.A., Casnellie, 

J.E., and Greengard, P. (1982) 

Nature 299, 734–736. 

31. Armstrong, E.G. et al. (1984) J. Clin. 

Endocrinol. Metab. 59, 867–874. 

32. Gilmour, D.S. and Lis, J.T. (1985) 

Mol. Cell. Biol. 5, 2009–2018. 

33. US Patent No. 4,604,365 

34. McCafferty, J., Griffiths, A.D., 

Winter, D., and Chiswell, D.J. 

(1990) Nature 348, 552–554. 

35. Spieker-Polet, H., Sethupathi, P., 

Yam, P.C., and Knight, K.L. (1995) 

Proc. Natl. Acad. Sci. USA 92, 

9348–9352. 

36. Reviewed in Taneja, S.S. (2004) 

Rev. Urol. 6, 19–28. 

37. Reviewed in Demidem, A. et al. 

(1997) Cancer Biother. Radiopharm. 

12, 177–186. 

38. Tolner, B. (2014) Therapeutic 

monoclonal antibodies approved or 

in review in the European Union or 

United States. The Antibody Society 

www.antibodysociety.org/news/approved_mabs.php. 

July 29, 2014. 

39. www.cellsignal.com/products/ 

primary-antibodies/9611 

40. Green, L.L. (1999) J. Immunol. 

Methods 10, 11–23. 

41. US Patent No. 6,441,140 

42. www.fda.gov/newsevents/newsroom/pressannouncements/2004/ 

ucm108244.htm 

43. www.cellsignal.com/XMT 

44. Cheung, W.C. et al. (2012) 

Nat. Biotechnol. 30, 447–452. 

45. www.fda.gov/newsevents/ 

newsroom/pressannouncements/ 

ucm340704.htm 

46. www.fda.gov/newsevents/ 

newsroom/pressannouncements/ 

ucm1193237.htm 

17

I 

Research 

Areas 

We are now at a dynamic and exciting phase of cell biology research. 

In the pages to follow, we hope to capture this excitement by providing 

our Research Area pages. Each Research Area includes overview 

text, images, tables, and reference pathway illustrations that provide 

a snapshot of information reflecting the latest thinking in the research 

community. The information contained within is a collaborative effort 

that represents contributions of both expert CST scientists and external 

reviewers from the global research community at the forefront of 

their respective fields. 

Research Areas are categorized into chapters covering the central 

themes of cell biology, so individual areas can be seen within their 

broader biological context. 

Section Includes: 

Introductory backgrounds and data features 

Reference pathways and tables 

Target lists for CST antibody products 

Citations for select targets 

Mitochondria 

Molecular landscape portraying the crosstalk between cellular metabolism (including 

proteins involved in the Warburg effect), mitochondrial transport, and apoptotic signaling. 

www.cellsignal.com/cstlandscapes 

19

01 

Section I: Research Areas 

Gene Expression, Epigenetics, 

and Nuclear Function 

Chromatin/Epigenetic Regulation 

The nucleosome, made up of four histone proteins (H2A, H2B, H3, and H4) and 147 bp of DNA, is 

the primary building block of chromatin. In addition to the core histone proteins, a number of histone 

variants exist (H2AX, H2AZ, MacroH2A, H3.3, and CENP-A) that confer different structural properties 

to nucleosomes and function in DNA repair, chromosome segregation during mitosis, and regulation 

of transcription. Histones were originally thought to function as a static scaffold for DNA packaging; 

however, they are now known to be dynamic proteins, undergoing multiple types of post-translational 

modifications (PTMs) and interacting with regulatory proteins to control gene expression in a highly 

regulated manner. 

Chromatin Modifying Enzymes 

Histone Acetylases and Deacetylases 

Protein acetylation plays a crucial role in regulating chromatin structure and transcriptional activity. 

Acetylation marks occur on lysine residues and are applied by histone acetyltransferases (HATs) and 

removed by histone deacetylases (HDACs). Acetylation within the histone tail weakens histone-DNA 

and histone-histone interactions and creates an open chromatin conformation that increases transcription 

factor access to DNA, while histone deacetylation limits gene activity by creating a closed 

conformation that limits transcription factor binding. In addition, acetylation creates binding sites for 

bromodomain-containing chromatin regulatory proteins. For example, the bromodomain-containing 

protein 4 (BRD4) is a chromatin-binding protein with a preference for acetyl-lysine 14 on histone H3 as 

well as acetyl-lysine 5 and acetyl-lysine 12 on histone H4. 

BRD4 binds to acetylated lysine residues within active chromatin regions. 

BRD4 (E2A7X) Rabbit mAb #13440: Chromatin IPs were performed with 

cross-linked chromatin from 4 x 10 6 MV-4-11 cells and either 10 µl of 

#13440 or 2 µl of Normal Rabbit IgG #2729, using SimpleChIP ® Enzymatic 

Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified 

by real-time PCR using SimpleChIP ® Human Bcl-2 Promoter Primers #12924, 

human c-Myc intron 1 primers, and SimpleChIP ® Human α Satellite Repeat 

Primers #4486. The amount of immunoprecipitated DNA in each sample is 

represented as a percent of the total input chromatin. 

BRD4 (E2A7X) 

Rabbit mAb #13440 

Normal Rabbit 

IgG #2729 

SirT6, a class III histone deacetylase, is 

expressed in wild-type but not knock-out MEFs. 

SirT6 (D8D12) Rabbit mAb #12486: WB analysis of extracts from SirT6 wild-type 

(WT) and knockout (KO) mouse embryonic fibroblasts (MEF) using #12486 (upper) 

or β-Actin (D6A8) Rabbit mAb #8457 (lower). SirT6 WT and KO MEF were kindly 

provided by Dr. David Lombard, University of Michigan. 

% of total input chromatin 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0 

Bcl-2 

Lanes 

1. SirT6 WT MEF 

2. SirT6 KO MEF 

c-Myc 

kDa 

60 

50 

40 

30 

α Satellite 

1 2 

SirT6 

chapter 01: GENE EXPRESSION, EPIGENETICS, AND NUCLEAR FUNCTION 

Histone Methyltransferases and Demethylases 

The histone methylation state determines active and inactive regions of the genome and is crucial for 

proper developmental programming. Methylation marks occur at lysine and arginine residues and are 

carried out by histone methyltransferases, including lysine methyltransferases EZH2, G9a, SUV39H1, 

and arginine methyltransferases PRMT1, PRMT4/CARM1, and PRMT5. Methylated histone residues 

are found within all four core histone proteins and are implicated in both transcriptional activation and 

silencing. Methylation facilitates binding of chromatin regulatory proteins (readers) that contain various 

methyl-lysine or methyl-arginine binding domains (PHD, chromo, WD40, Tudor, MBT, Ankyrin repeats, 

PWWP domains). Methylation marks are removed by demethylases such as Jumonji C domain family 

proteins (JARID, JMJD, UTX, UTY, FBXL10, FBXL11) and LSD1. 

A 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

20 

1 

2 3 

Ezh2 

B 

C 

1.6 

1.4 

1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0 

Ezh2 (D2C9) XP ® Rabbit mAb #5246: WB analysis of extracts from MCF7, Neuro-2a, and COS-7 cell lines (A) using #5246. IHC analysis 

of paraffin-embedded human lymphoma (B) using #5246. Chromatin IPs were performed with cross-linked chromatin from 4 x 10 6 NCCIT 

cells (C) and either 5 µl of #5246 or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) 

#9003. The enriched DNA was quantified by real-time PCR using SimpleChIP ® Human HoxA1 Intron 1 Primers #7707, SimpleChIP ® Human 

HoxA2 Promoter Primers #5517, and SimpleChIP ® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in 

each sample is represented as a percent of the total input chromatin. 


HoxA1 

HoxA2 

α Satellite 

Other Modifications: Phosphorylation, 

Ubiquitination, DNA Methylation 

Chromatin structure is also modulated through other PTMs such as phosphorylation and ubiquitination 

of histone proteins, which affect association with DNA-interacting proteins and have been recently 

identified to play a role in coordinating other histone modifications. For example, histone H2B is 

ubiquitinated at Lys120, which is associated with the transcribed region of active genes. Ubiquitination 

of H2B at Lys120 is essential for subsequent methylation of histone H3 Lys4 and 79, two additional 

histone modifications that regulate transcriptional initiation and elongation. 

In addition, methylation of DNA at cytosine residues affects chromatin folding by recruiting methyl-DNA 

binding proteins (MeCP2, MBD), which recruit additional chromatin modifying complexes and inhibit 

DNA binding of methylation-sensitive transcriptional activators. DNA methylation is critical for proper 

regulation of gene silencing, genomic imprinting, and development. Two families of mammalian DNA 

methyltransferases have been identified, DNMT1 and DNMT3, which play distinct roles in embryonic 

stem cells and adult somatic cells. Activity of these enzymes is regulated by accessory proteins such 

as DMAP1 and UHRF1, which target the associated DNMT to mediate proper methylation patterns to 

newly synthesized DNA during replication. DNA methylation changes are frequently associated with 

cancer; general hypomethylation of the genome and localized hypermethylation of CpG islands within 

tumor suppressor promoter regions can both be found during various stages of cancer development. 

Ezh2, a member of 

the Polycomb Group 

proteins, is expressed 

in various cell lines and 

cancers, and associates 

with HoxA genes. 

Lanes 

1. MCF7 

2. Neuro-2a 

3. COS-7 

Ezh2 (D2C9) XP ® 


Normal Rabbit 

IgG #2729 

Chromatin/Epigenetics Resources 

Please visit our website for additional resources and products relating to the study of Chromatin/Epigenetics. 

www.cellsignal.com/cstchromatin 

60 

50 

40 

30 

β-Actin 

Ubiquityl-Histone H2B (Lys120) (D11) XP ® Rabbit mAb #5546: 

Chromatin IPs were performed with cross-linked chromatin from 4 x 10 6 

HeLa cells and either 10 μl of #5546 or 2 μl of Normal Rabbit IgG #2729 

using SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. 

The enriched DNA was quantified by real-time PCR using SimpleChIP ® 

Human γ-Actin Promoter Primers #5037, SimpleChIP ® Human γ-Actin Intron 

3 Primers #5047, SimpleChIP ® Human GAPDH Promoter Primers #4471, 

and SimpleChIP ® Human GAPDH Intron 2 Primers #4478. The amount of 

immunoprecipitated DNA in each sample is represented as a percent of the 

total input chromatin. 

Ubiquityl-Histone H2B (Lys120) 

(D11) XP ® Rabbit mAb #5546 

Normal Rabbit 

IgG #2729 


1.2 

1.0 

0.8 

0.6 

0.4 

0.2 

0 

γ-Actin 

Promoter 

γ-Actin 

Intron 3 

GAPDH 

Promoter 

GAPDH 

Intron 2 

Ubiquitination of 

histone H2B at Lys120 

is associated with the 

transcribed region of 

active genes. 

20 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys. 


21



ATP-dependent Chromatin Remodeling Proteins 

ATP-dependent remodeling complexes, such as SWI/SNF, NuRD, and ISWI, make structural changes 

to chromatin by using their ATPase catalytic subunit to disrupt histone-DNA contacts and reposition 

nucleosomes in order to expose regions of DNA to critical regulatory proteins necessary for transcription, 

DNA replication, and repair. 

SWI/SNF Complex 

The SWI/SNF complex (BAF and PBAF complexes in humans) consists of multiple subunits and contains 

either a BRM (SMARCA2) or a BRG1 (SMARCA4) protein that acts as an ATPase. Components of the 

SWI/SNF complex are commonly mutated in cancer and are the focus of many research efforts as 

potential therapeutic targets. 

BAF Complex 

ACTL6A/B 

SMARCC1 

SMARCA 

2/4 

SMARCC2 

ARID1A/B 

Polycomb Group Proteins 

Polycomb group (PcG) proteins help maintain cell identity, stem cell self-renewal, cell cycle regulation, 

and oncogenesis by silencing genes that promote cell lineage specification, cell death, and cell cycle 

arrest. PcG proteins exist in two complexes: PRC2 (EED-EZH2), which methylates histone H3 on Lys27 

(H3K27), and the PRC1 complex, which ubiquitinylates histone H2A on Lys119 in response to H3K27 

methylation. 

Canonical PRC1 

PHC 

PCGF2/4 

RING1A/B 

CBX 

Blackledge, N.P., et al. (2014) Cell 157, 1445−1459. 

Variant PRC1 

PCGF1-6 

RING1A/B 

rybp/ 

yaf2 

PRC2 Complex 

SUZ12 

EZH2 

EED 

RBAP46/48 

AEBP2 

Jarid2 

PBAF Complex 

SMARCD 

1/2/3 

ACTL6A/B 

SMARCB1 

SMARCA 

2/4 

SMARCE1 

DPF1/2/3 

ARID2 

PBRM1 

Wang, X., et al. (2014) 

Clin. Cancer Res. 20, 21−27. 

BRD7 

Bmi1 (D20B7) XP ® Rabbit mAb #6964: Chromatin IPs were performed with 

cross-linked chromatin from 4 x 10 6 NCCIT cells and either 10 µl of #6964 or 

2 µl of Normal Rabbit IgG #2729 using SimpleChIP ® Enzymatic Chromatin IP 

Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time 

PCR using SimpleChIP ® Human HoxA1 Intron 1 Primers #7707, SimpleChIP ® 

Human HoxA2 Promoter Primers #5517, and SimpleChIP ® Human α Satellite 

Repeat Primers #4486. The amount of immunoprecipitated DNA in each 

sample is represented as a percent of the total input chromatin. 

Bmi1 (D20B7) XP ® 


Normal Rabbit 

IgG #2729 


0.5 

0.4 

0.3 

0.2 

0.1 

0 

HoxA1 

HoxA2 

α Satellite 

Bmi1 (PCGF4), a 

component of the 

PRC1 complex of 

polycomb group proteins, 

associates with 

silenced Hox genes. 

SMARCC1 

SMARCC2 

SMARCD 

1/2/3 

SMARCB1 

SMARCE1 

DPF1/2/3 

BRM, one of two ATPase catalytic subunits for the SWI/SNF complex, 

associates with estrogen-responsive genes in cells treated with β-estradiol. 

BRM (D9E8B) XP ® Rabbit mAb #11966: Chromatin IPs were performed 

with cross-linked chromatin from 4 x 10 6 MCF7 cells grown in phenol red-free 

medium and 5% charcoal-stripped FBS for 4 d followed by treatment with 

β-estradiol (10 nM, 45 min) and either 5 μl of #11966 or 2 μl of Normal 

Rabbit IgG #2729 using SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic 

Beads) #9003. The enriched DNA was quantified by real-time PCR using 

SimpleChIP ® Human ESR1 Promoter Primers #9673, SimpleChIP ® Human 

pS2 Promoter Primers #9702, and SimpleChIP ® Human α Satellite Repeat 

Primers #4486. The amount of immunoprecipitated DNA in each sample is 

represented as a percent of the total input chromatin. 

BRM (D9E8B) XP ® 


Normal Rabbit 

IgG #2729 

NuRD Complex 

The transcriptional repressor nucleosome remodeling and histone deacetylase (NuRD) complex is 

composed of multiple subunits, including histone deacetylases (HDAC1 and HDAC2) and the ATPdependent 

helicases CHD3 and CHD4. The NuRD complex plays an important role in regulating genes 

responsible for embryonic stem cell pluripotency and differentiation. 

ISWI Complex 

The mammalian imitation SWI (ISWI) chromatin remodeling complexes have been shown to alter 

nucleosome spacing in vitro. ISWI complexes are characterized by two ATPase subunits: SNF2H and 

SMARCA1 (SNF2L). SNF2H is part of several chromatin-remodeling complexes, including ACF1, RSF1, 

CHRAC, NoRC, WSTF, and WCRF180, and is important for regulation of chromatin structure, DNA 

damage response, and development. SMARCA1 is the catalytic subunit of the nucleosome remodeling 

factor (NURF) complex and plays an important role in neuronal and T cell development. 


0.30 

0.25 

0.20 

0.15 

0.10 

0.05 

0 

ESR1 

pS2 

α Satellite 

RNA Polymerase II Modifications 

RNA polymerase II (RNAPII) is a large multiprotein complex that functions as a DNA-dependent RNA 

polymerase, catalyzing the transcription of DNA into RNA. The largest subunit, RNAPII subunit B1 

(Rpb1), also known as RNAPII subunit A (POLR2A), contains a unique heptapeptide sequence (Tyr1, 

Ser2, Pro3, Thr4, Ser5, Pro6, Ser7), which is repeated up to 52 times in the carboxy-terminal domain 

(CTD) of the protein. This CTD heptapeptide repeat is subject to multiple PTMs, which dictate the 

functional state of the polymerase complex. Phosphorylation of the CTD during the active transcription 

cycle integrates transcription with chromatin remodeling and nascent RNA processing by regulating the 

recruitment of chromatin modifying enzymes and RNA processing proteins to the transcribed gene. 

Phospho-Rpb1 CTD (Ser2/Ser5) (D1G3K) Rabbit mAb # 13546: 

Chromatin IPs were performed with cross-linked chromatin from 4 x 10 6 

HeLa cells and either 10 μl of #13546 or 2 μl of Normal Rabbit IgG #2729 

using SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. 

The enriched DNA was quantified by real-time PCR using SimpleChIP ® 

Human β-Actin Promoter Primers #13653, human β-Actin intron 1 primers, 

SimpleChIP ® Human β-Actin 3ʹ UTR Primers #13669, and SimpleChIP ® 

Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated 

DNA in each sample is represented as a percent of the total input chromatin. 

Phospho-Rpb1 CTD (Ser2/Ser5) 

(D1G3K) Rabbit mAb #13546 

Normal Rabbit 

IgG #2729 


4.0 

3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0 

β-Actin 

Promoter 

β-Actin 

Intron 1 

β-Actin 

3’UTR 

α Satellite 

Select Reviews 

Del Rizzo, P.A. and Trievel, R.C. (2014) Biochim. Biophys. Acta. Jun 17 [Epub ahead of print] • Kurdistani, S.K. (2014) 

Curr. Opin. Genet. Dev. 26, 53−58. • Marmorstein, R. and Zhou, M.M. (2014) Cold Spring Harb. Perspect. Biol. 6, a018762. 

• Napolitano, G., Lania, L., and Majello, B. (2014) J. Cell Physiol. 229, 538−544. • Narlikar, G.J., Sundaramoorthy, R., and 

Owen-Hughes, T. (2013) Cell 154, 490−503. • Sanchez, R., Meslamani, J., and Zhou, M.M. (2014) Biochim. Biophys. Acta. 

1839, 676−685. • Schwartz, Y.B. and Pirrotta, V. (2013) Nat. Rev. Genet. 14, 853−864. • Swygert, S.G. and Peterson, C.L. 

(2014) Biochim. Biophys. Acta. 1839, 728−736. • Van Rechem, C. and Whetstine, J.R. (2014) Biochim. Biophys. Acta. May 

23 [Epub ahead of print] 

Phospho-Rpb1 CTD 

(Ser2/Ser5) is associated 

with active gene regions. 



23



These protein targets represent key 

nodes within chromatin/epigenetic 

regulation signaling pathways and 

are commonly studied in chromatin/ 

epigenetic regulation research. Primary 

antibodies, antibody conjugates, and 

antibody sampler kits containing these 

targets are available from CST. 

Listing as of September 2014. See our 

website for current product information. 

M Monoclonal Antibody 

P Polyclonal Antibody 

E PathScan ® ELISA Kits 

S SignalSilence ® siRNA 

C Antibody Conjugate 

Commonly Studied Chromatin/Epigenetic Regulation Targets 

Target M P E S C 

ACF1 

• 

ARID1A/BAF250A 

• • 

ASF1A 

• 

ASF1B 

• • 

ASH2L 

• 

Bmi1 • • • 

BORIS 

• 

BTAF1 

• 

Brd2 

• 

BRD4 

• 

Brg1 

• 

BRM 

• • 

CABIN1 

• 

CBP 

• 

Acetyl-CBP (Lys1535)/p300 (Lys1499) • 

CDK7 

• • 

CDK8 

• 

CDK9 

• 

Phospho-CDK9 (Thr186) • 

CDK12 

• 

CENP-A 

• • 

Phospho-CENPA (Ser7) • 

CHAF1A 

• 

CHD1 • • 

CHD1L 

• 

CHD2 

• 

CHD3 

• 

CHD4 

• • 

CHD7 

• 

CHD8 

• • 

CLOCK 

• 

CTCF • • • 

CTDSPL2 

• 

Phospho-CTDSPL2 (Ser104) • 

CTR9 

• 

CXXC1 

• 

Cyclin T1 

• 

DBC1 

• • 

Phospho-DBC1 (Thr454) • 

DMAP1 

• 

DNMT1 

• 

DNMT3A 

• • 

DNMT3L 

• 

DR1 

• 

ELP1/IKBKAP 

• 

ELP3 

• 

ESET 

• 

EWS • • 

Ezh2 

• • • • • 

G9a/EHMT2 

• 

GCN5L2 

• 

Histone H2A 

• • 

Acetyl-Histone H2A (pan) 

• 

Acetyl-Histone H2A (Lys5) • 

Ubiquityl-Histone H2A (Lys119) • 

MacroH2A1 

• • 

MacroH2A1.2 

• 

Histone H2A.X 

• • 


Phospho-Histone H2A.X (Ser139) • • • 

• 

Phospho-Histone H2A.X (Ser139/ 

Tyr142) 

Histone H2A.Z 

• 

Histone H2B • • • • 

Acetyl-Histone H2B (Lys5) • • 


Acetyl-Histone H2B (Lys15) • 


Phospho-Histone H2B (Ser14) • 

Ubiquityl-Histone H2B (Lys120) • 

Histone H3 • • • • 

Acetylated Histone H3 (pan) • 

Acetyl-Histone H3 (Lys9) • • • 

• 

Acetyl- and Phospho-Histone H3 

(Lys9/Ser10) 

Acetyl-Histone H3 (Lys14) • 


Acetyl-Histone H3 (Lys27) • • 



Methyl-Histone H3 (Arg2) • 

Mono-Methyl-Histone H3 (Lys4) • • • 

Di-Methyl-Histone H3 (Lys4) • • 

Tri-Methyl-Histone H3 (Lys4) • • • • 

• 

Symmetric Di-Methyl-Histone H3 

(Arg8) 

Pan-Methyl-Histone H3 (Lys9) • • 

Di-Methyl-Histone H3 (Lys9) • • • 

Di/Tri-Methyl-Histone H3 (Lys9) • 

Tri-Methyl-Histone H3 (Lys9) • 


Tri-Methyl-Histone H3 (Lys27) • • • 


Tri-Methyl-Histone H3 (Lys36) • • 

Mono-Methyl-Histone H3 (Lys79) • • 

Di-Methyl-Histone H3 (Lys79) • 


Cleaved Histone H3 (Thr22) • • 

Phospho-Histone H3 (Thr3) • • 

Phospho-Histone H3 (Ser10) • • • • 

Phospho-Histone H3 (Thr11) • • 

Phospho-Histone H3 (Ser28) • 

Histone H4 • • • 

Acetyl-Histone H4 (pan) 

• 





Mono-Methyl-Histone H4 (Lys20) • 

Di-Methyl-Histone H4 (Lys20) • 


HDAC1 

• • 

HDAC2 

• • 

HDAC3 

• • 

Phospho-HDAC3 (Ser424) • 

HDAC4 • • • 

• 

Phospho-HDAC4 (Ser246)/HDAC5 

(Ser259)/HDAC7 (Ser155) 


Phospho-HDAC4 (Ser632)/HDAC5 

(Ser661)/HDAC7 (Ser486) • 

HDAC6 

• 

HELLS 

• 

HEXIM1 

• • 

HIRA 

• 

HMGA1 

• 

HMGA2 

• • 

HMGB1 

• • 

HMGB2 

• 

HMGN1 

• • 

HMGN2 

• 

HP1α 

• 

HP1α/β 

• 

HP1β 

• • 

HP1γ 

• 

Phospho-HP1γ (Ser83) • 

INTS9 

• 

JARID1A 

• 

JARID1B 

• 

JARID1C 

• 

JARID2 

• 

JMJD1B/JHDM2B 

• 

JMJD1B 

• 

JMJD2A 

• 

JMJD2B • • • 

JMJD3 

• 

LCMT1 

• 

LEDGF 

• 

LSD1 

• • 

MBD3 

• 

MeCP2 

• 

MED12 

• 

MED23 

• 

MED26 

• 

Menin 

• 

MEP50 

• • 

MLLT1/ENL 

• 

MORF4L1/MRG15 • • 

MTA1 

• 

NCoR1 

• 

NFI-C 

• 

Nucleomethylin 

• 

NRF2 

• 

Nucleolin 

• 

NUT1 

• 

PAF1 

• 

PCAF 

• 

PHC1 

• 

PHF2 

• 

PHF20 

• 

POLR3A 

• 

Pontin/RUVBL1 

• 

PRMT1 

• 

PRMT4/CARM1 

• • 

PRMT5/Skb1Hs Methyltransferase • 

RAD21 

• 

RBAP46/RBAP48 

• • 

RBBP5 

• • 

RBAP46 

• 


Reptin/RuvBL2 

• • 

Ring1A 

• • 

RING1B 

• 

RNF20 

• 

RNF40 

• • 

Rpb1 CTD 

• 

Phospho-Rpb1 CTD (Ser2) • 

Phospho-Rpb1 CTD (Ser2/Ser5) • 

Phospho-Rpb1 CTD (Ser5) • • 

Phospho-Rpb1 CTD (Ser7) • 

SATB1 

• • 

Phospho-SATB1 (Ser47) • 

SET7/SET9 

• • 

SET8 

• 

SIN3A 

• 

SirT1 • • • 

Phospho-SirT1 (Ser27) • 

Phospho-SirT1 (Ser47) • 

SirT2 

• 

SirT3 

• 

SirT5 

• 

SirT6 • • • 

SirT7 

• 

SMARCA1 

• • 

SMARCAD1 

• 

SMARCC1/BAF155 • • 

SMARCC2/BAF170 • • 

SMYD2 

• • 

SMYD3 

• 

SNF2H 

• 

SNF5 

• 

SP1 • • • 

SPT5 

• 

SPT16 

• 

SRC-1 

• 

SRC-3 

• 

Phospho-SRC-3 (Thr24) • 

SSRP1 

• • 

SSU72 

• 

SUV39H1 

• 

SUZ12 

• 

TAF1 

• 

TAF15 

• 

TBP 

• • 

TCEB3/Elongin A 

• 

TFII-I 

• 

TFIIB 

• 

TFIIFα 

• 

TH1L 

• • 

Tip60 

• 

Topoisomerase IIα 

• • 

Phospho-Topoisomerase IIα (Ser1469) • 

TRIM29/ATDC 

• 

TRRAP 

• 

UHRF1 

• 

WDR5 

• 

WSTF 

• 

XPB 

• 

XPD 

• 

YY1 

• 

155 

2012–2014 citations 

CST antibodies for Histone H3 have 

been cited over 155 times in highimpact, 

peer-reviewed publications 

from the global research community. 

Select Citations: 

Crotti, A. et al. (2014) Mutant 

Huntingtin promotes autonomous 

microglia activation via myeloid 

lineage-determining factors. Nat. 

Neurosci. 17, 513−521. 

Tanaka-Nakanishi, A. et al. (2014) 

HTLV-1 bZIP factor suppresses 

apoptosis by attenuating the function 

of FoxO3a and altering its localization. 

Cancer Res. 74, 188−200. 

Hast, B.E. et al. (2014) Cancerderived 

mutations in KEAP1 impair 

NRF2 degradation but not ubiquitination. 

Cancer Res. 74, 808−817. 

Chen, C.H. et al. (2014) Synergistic 

interaction between the HDAC inhibitor, 

MPT0E028, and sorafenib in liver 

cancer cells in vitro and in vivo. Clin. 

Cancer Res. 20, 1274−1287. 

Ghare, S.S. et al. (2014) Coordinated 

Histone H3 Methylation and Acetylation 

Regulate Physiologic and Pathologic 

Fas Ligand Gene Expression in 

Human CD4+ T Cells. J. Immunol. 

193, 412−421. 

Karijolich, J. et al. (2014) Coordinated 

Histone H3 Methylation and 

Acetylation Regulate Physiologic and 

Pathologic Fas Ligand Gene Expression 

in Human CD4+ T Cells. J. Virol. 

88, 7024−7035. 

Galli, G.G. et al. (2014) Prdm5 

suppresses Apc(Min)-driven intestinal 

adenomas and regulates monoacylglycerol 

lipase expression. Oncogene 

33, 3342−3350. 

Khalaj, M. et al. (2014) A missense 

mutation in Rev7 disrupts formation of 

Polzeta, impairing mouse development 

and repair of genotoxic agent-induced 

DNA lesions. J. Biol. Chem. 289, 

3811−3824. 

Belozerov, V.E. et al. (2014) In vivo 

interaction proteomics reveal a novel 

p38 mitogen-activated protein kinase/ 

Rack1 pathway regulating proteostasis 

in Drosophila muscle. Mol. Cell Biol. 

34, 474−484. 

Gray, C.M. et al. (2014) Noncanonical 

NF-kappaB signaling is limited by 

classical NF-kappaB activity. Sci. 

Signal. 7, 13. 


www.cellsignal.com/cstchromatin 25


Protein Acetylation 

Histone 

Acetylation 

Protein 

Acetylation 

GCN5L2 

Cell Cycle 

Rb 

Stability 

DNA Damage 

SRC-3 

Tip60 

ATM 

DNA 

Repair 

TF 

CBP/ 

p300 

TF 

HDAC Classes 

Class I: HDAC1-3, 8 

Class II: HDAC4-7, 9, 10 

Class III: SIRT1-7 

Class IV: HDAC11 

p53 

TF 

Stability 

Acetylases 

PCAF 

Su 

Sirtuins 

Regulatory 

Signaling Pathways 

DNA 

Damage 

AceCS1 

PEPCK 

PGC-1α 

Idh2 

TFIID 

TFIIA TBP 

HDAC 

NuRD 

NcoR 

Sin3 


Tip60 

Histones 

Signaling Pathways 

Metabolism 

Deacetylases 

SirT1 

Tip60 

SirT1 

SirT3 

Cytoskeletal Regulation Ribosomal Proteins 

Chaperones 

BRCA1 

TATA 

RNA Splicing 

Acetylation 

Acetyl 

Transferases 

ATF-2 MORF 

CBP MOZ 

CDY p300 

PCAF CLOCK PCAF 

CBP/ 

p300 

EWI p/CIP 

Su 

Elp3 SRC-1 

SRC-3 

GCN5L2 SRC-3 

GRIP hTAF II 

250 

α-Importin 

HAT1 TFIIB 

HBO1 Tip60 

MCM3AP 

Nuclear Transport 

Transcription 

TFIIH 

Pluripotency 

TFIIF 

Development 

TFIIB 

RNA Pol II Differentiation of 

Neural Stem Cells 

TFIIE 

BMP, Wnt, Notch 

AceCS1 

PEPCK 

PGC-1α 

Idh2 

Deacetylation 

Fatty Acid Synthesis 

Histone Acetylation 

Gluconeogenesis 

Expression of 

Gluconeogenic Genes 

NADPH 

Lysine acetylation is a reversible post-translational modification that plays a crucial role in regulating protein function, chromatin structure, and gene expression. Many 

transcriptional coactivators possess intrinsic acetylase activity, while transcriptional corepressors are associated with deacetylase activity. Acetylation complexes (such as CBP/ 

p300 and PCAF) or deacetylation complexes (such as Sin3, NuRD, NcoR, and SMRT) are recruited to DNA-bound transcription factors (TFs) in response to signaling pathways. 

Histone hyperacetylation by histone acetyltransferases (HATs) is associated with transcriptional activation, whereas histone deacetylation by histone deacetylases (HDACs) is 

associated with transcriptional repression. Histone acetylation stimulates transcription by remodeling higher order chromatin structure, weakening histone-DNA interactions, 

and providing binding sites for transcriptional activation complexes containing proteins that possess bromodomains, which bind acetylated lysine. Histone deacetylation 

represses transcription through an inverse mechanism involving the assembly of compact higher order chromatin and the exclusion of bromodomain-containing transcription 

activation complexes. Histone hypoacetylation is a hallmark of silent heterochromatin. Site-specific acetylation of a growing number of non-histone proteins has been shown 

to regulate their activity, localization, specific interactions, and stability/degradation. Remarkably, recent advances in mass spectrometry technologies allowed high resolution 

mapping of most of the acetylation sites in all the proteome. These studies demonstrated that the “acetylome” encompasses nearly ~3600 acetylation sites in roughly ~1750 

proteins, suggesting that this modification is one of the most abundant chemical modifications in nature. Indeed, it appears that this mark can influence the activity of proteins 

in diverse biological processes, including chromatin remodeling, cell cycle, splicing, nuclear transport, mitochondrial biology, and actin nucleation. At an organismal level, 

acetylation plays an important role in immunity, circadian rhythmicity, and memory formation. Protein acetylation is becoming a favorable target in drug design for numerous 

disease conditions. 

Select Reviews: 

Albaugh, B.N., Arnold, K.M., and Denu, J.M. (2011) Chem. Bio. Chem. 12, 290–298. • Choudhary, C., Kumar, C., Gnad, F., Nielsen, M.L., Rehman, M., Walther, T.C., 

Olsen, J.V., and Mann, M. (2009) Science 325, 834–840. • Dali-Youcef, N., Lagouge, M., Froelich, S. et al. (2007) Ann. Med. 39, 335–345. • Finkel, T., Deng, C.H., and 

Mostoslavsky, R. (2009) Nature 460, 587–591. • Haberland, M., Montgomery, R.L., and Olson, E.N. (2009) Nat. Rev. Genet. 10, 32–42. • Peng, L. and Seto, E. (2011) 

Handbook Exp. Pharmac. 206, 39–56. • Spange, S., Wagner, T., Heinzel, T., and Krämer, O.H. (2009) Int. J. Biochem. Cell Biol. 41, 185–198. • Yang, X.J. and Seto, E. 

(2007) Oncogene 26, 5310–5318. • Yang, X.J. and Seto, E. (2008) Mol. Cell 31, 449–461. 

© 2002–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Raul Mostoslavsky, Harvard Medical School, Boston, MA, for reviewing this diagram. 

H4K20me 

Methylases: 

SET8/KMT5A (+) 

NSD1/KMT3B (+,†) 

ASH1L/KMT2H (+,†,‡) 

SUV420H1/KMT5B (†,‡) 

SUV420H2/KMT5C (†,‡) 

NSD2/KMT3G (+,†) 

Demethylases: 

JHDM1D/KDM7A (+,†) 

PHF8/KDM7B (+,†) 

H3K9me 

Methylases: 

PRDM3/KMT8E (+) 

PPRDM16/KMT8F (+) 

G9a/EHMT2/KMT1C (+,†) 

EHMT1/KMT1D (+,†) 


PRDM2/KMT8A (+,†,‡) 

PRDM8/KMT8D (†) 

SUV39H1/KMT1A (†,‡) 

SUV39H2/KMT1B (†,‡) 

ESET/KMT1E (†,‡) 

CLLD8/KMT1F (†,‡) 

Demethylases: 

AOF1/KDM1B (+,†) 

JMJD1A/KDM3A (+,†) 

JMJD1B/KDM3B (+,†) 

JMJD1C/KDM3C (+,†) 



JMJD2A/KDM4B (†,‡) 

JMJD2B/KDM4B (†,‡) 

JMJD2C/KDM4C (†,‡) 

JMJD2D/KDM4D (†,‡) 

KDM4E/KDM4DL (†,‡) 

H3K27me 

Methylases: 

Ezh2/KMT6 (+,†,‡) 

NSD2/KMT3G (+,†,‡) 

NSD3/KMT3F (†,‡) 

Demethylases: 



UTX/KDM6A (†,‡) 

JMJD3/KDM6B (†,‡) 

Histone Lysine Methylation 

SUV 

39h 

HP1 

Me 

Me 

Transcriptionally Inactive 

Chromatin 

SUV 

39h 

PRC1 

PC 

Me 

PRC1 

PC 

Me 

HP1 

Me 

Me 

SUV 

39h 

HP1 

Me 

Me 

SUV 

39h 

HP1 

Me 

Pericentric Heterochromatin 

Inactive X Chromosome 

Rb-Mediated Repression 

PRC1 

PC 

Me 

PRC1 

PC 

Me 

Me 

Pericentric Heterochromatin 

Inactive X Chromosome 

Hox Gene Repression 

Methylation 

H4K20me 

Demethylation 

Methylation 

H3K9me 

Demethylation 

Methylation Degree: + Mono † Di ‡ Tri 

Methylation 

H3K27me 

Demethylation 


Methylation 

Methylation 

Demethylation 

Methylation 

H3K79me 

Demethylation 

H3K4me 

Demethylation 

H3K36me 

Transcriptionally Active 

Chromatin 

NURF 

BPTF 

Me 

Me 

Me 

Me 

NURF 

BPTF 

Me 

Me 

Me 

Me 

Me 

Anti-Silencing 

Me 

Me 

Transcription Initiation 

Me 

Transcription Elongation 

The nucleosome is the primary building block of chromatin containing a histone octamer composed of two sets of H3-H4 and H2A-H2B dimers. 

Originally thought to function as a static scaffold for DNA packaging, histones have more recently been shown to be dynamic proteins, undergoing 

multiple types of post-translational modifications and impacting numerous nuclear functions. Lysine methylation is one such modification and is 

a major determinant for genome organization and the formation of active and inactive regions of the genome. Lysines can have three different 

methylation states (mono-, di-, and tri-) that are associated with different nuclear features and transcriptional states. In order to establish these 

methylation states, cells have enzymes that both add (lysine methyltransferases- KMTs) and remove (lysine demethylases- KDMs) different degrees 

of methylation from specific lysines within the histones. To date, all but one histone lysine methyltransferase (DOT1L/KMT4) has a conserved 

catalytic SET domain that was originally identified in the Drosophila Su[var]3-9, Enhancer of zeste, and Trithorax proteins. In the case of the histone 

lysine demethylases, there are two different classes: the FAD-dependent amine oxidases and the JmjC-containing enzymes. Both KMTs and KDMs 

have specificity for specific lysine residues and degrees of methylation within the histone tails. Therefore, all KMTs and KDMs are not the same in 

their biological functions or roles in transcriptional output. 

Lysine methylation has been implicated in both transcriptional activation (H3K4, K36, K79) and silencing (H3K9, K27, H4K20). The degree of 

methylation is associated with different outcomes. For example, H4K20 monomethyation (H4K20me1) is observed in the bodies of active genes, 

while H4K20 trimethylation (H4K20me3) is affiliated with gene repression and compacted genomic regions. Gene regulation is also affected by 

the location of the methylated lysine residue with respect to the DNA sequence. For example, H3K9me3 at promoters is associated with gene 

repression, while some induced genes have H3K9me3 in the gene body. Since this modification is uncharged and chemically inert, the impact 

these modifications have is through recognition by other proteins with binding motifs. Lysine methylation coordinates the recruitment of chromatin 

modifying enzymes. Chromodomains (e.g., found in HP1, PRC1), PHD fingers (e.g., found in BPTF, ING2, SMCX/KDM5C), Tudor domains (e.g., 

found in 53BP1 and JMJD2A/KDM4A), PWWP domains (e.g., found in ZMYND11) and WD-40 domains (e.g., found in WDR5) are among a 

growing list of methyl lysine binding modules found in histone acetyltransferases, deacetylases, methylases, demethylases and ATP-dependent 

chromatin remodeling enzymes. Lysine methylation provides a binding surface for these enzymes, which then regulate chromatin condensation 

and nucleosome mobility, active and inactive transcription as well as DNA repair and replication. In addition, lysine methylation can block binding 

of proteins that interact with unmethylated histones or directly inhibit catalysis of other regulatory modifications on neighboring residues. 

Histone methylation is crucial for proper programming of the genome during development and misregulation of the methylation machinery can 

lead to diseased states such as cancer. In fact, cancer genome analyses have uncovered lysine mutations in H3K27 and H3K36. These sites are 

enriched in subsets of cancer. Therefore, an entirely new therapeutic and biomarker space is emerging with the discovery of these enzymes, the 

impact modifications have on the genome and disease associated mutations. 


Black, J.C., Van Rechem, C., and Whetstine, J.R. (2012) Mol. Cell. 48, 491–507. • Greer, E.L. and Shi, Y. (2012) Nat. Rev. Genet. 13, 343–357. 

• Herz, H.M., Garruss, A., and Shilatifard, A. (2013) Trends Biochem. Sci. 38, 621–639. • Kooistra, S.M. and Helin, K. (2012) Nat. Rev. Mol. 

Cell Biol. 13, 297–311. • Tee, W.W. and Reinberg, D. (2014) Development 141, 2376–2390. • Van Rechem, C. and Whetstine, J.R. (2014) 

Biophys. Acta. May 23 [Epub ahead of print]. 

© 2006–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Jonathan Whetstine for reviewing this diagram. 

H3K4me H4K20me 

Methylases: Methylases: 

MLL1/KMT2A SET8/KMT5A (+,†,‡) (+) 

MLL2/KMT2B NSD1/KMT3B (+,†,‡) (+,†) 

MLL3/KMT2C ASH1L/KMT2H (+) (+,†,‡) 

MLL4/KMT2D (+) SUV420H1/KMT5B (†,‡) 

SET1A/KMT2F (+,†,‡) 

SUV420H2/KMT5C (†,‡) 

SET1B/KMT2G (+,†,‡) 

NSD3/KMT3F NSD2/KMT3G (+,†) (+,†) 

NSD2/KMT3G Demethylases: (+,†) 

SET7/KMT7 JHDM1D/KDM7A (†) (+,†) 

SMYD3/KMT3E PHF8/KDM7B (†,‡) (+,†) 


PRDM9/KMT8B H3K9me (‡) 

Demethylases: 

Methylases: 

LSD1/KDM1A (+,†) 

PRDM3/KMT8E (+) 


JARID1A/KDM5A PPRDM16/KMT8F (+) (†,‡) 

JARID1B/KDM5B G9a/EHMT2/KMT1C (†,‡) (+,†) 

JARID1C/KDM5C EHMT1/KMT1D (†,‡) (+,†) 

JARID1D/KDM5D ASH1L/KMT2H (†,‡) (+,†,‡) 

PRDM2/KMT8A (+,†,‡) 

PRDM8/KMT8D (†) 

H3K36me SUV39H1/KMT1A (†,‡) 

Methylases: 

SUV39H2/KMT1B (†,‡) 


SMYD2/KMT3C ESET/KMT1E (+,†) (†,‡) 

NSD2/KMT3G CLLD8/KMT1F (+,†) (†,‡) 

SET2/KMT3A Demethylases: (‡) 

Demethylases: AOF1/KDM1B (+,†) 

JMJD1A/KDM2A JMJD1A/KDM3A (+,†) (+,†) 

JMJD1B/KDM2B JMJD1B/KDM3B (+,†) (+,†) 

JMJD2A/KDM4A (†,‡) 

JMJD1C/KDM3C (+,†) 




JMJD2D/KDM4D PHF8/KDM7B (†,‡) (+,†) 

KDM4DL/KDM4E JMJD2A/KDM4B (†,‡) (†,‡) 




KDM4E/KDM4DL (†,‡) 

H3K79me 

Methylases: 

H3K27me 

DOTIL/KMT4 (+,†,‡) 

Demethylases: Methylases: 

Ezh2/KMT6 (+,†,‡) 

NSD2/KMT3G (+,†,‡) 

NSD3/KMT3F (†,‡) 

Demethylases: 



UTX/KDM6A (†,‡) 

JMJD3/KDM6B (†,‡) 

H3K4me 

Methylases: 

MLL1/KMT2A (+,†,‡) 

MLL2/KMT2B (+,†,‡) 

MLL3/KMT2C (+) 

MLL4/KMT2D (+) 

SET1A/KMT2F (+,†,‡) 

SET1B/KMT2G (+,†,‡) 

NSD3/KMT3F (+,†) 


SET7/KMT7 (†) 

SMYD3/KMT3E (†,‡) 


PRDM9/KMT8B (‡) 

Demethylases: 

LSD1/KDM1A (+,†) 


JARID1A/KDM5A (†,‡) 

JARID1B/KDM5B (†,‡) 

JARID1C/KDM5C (†,‡) 

JARID1D/KDM5D (†,‡) 

H3K36me 

Methylases: 


SMYD2/KMT3C (+,†) 


SET2/KMT3A (‡) 

Demethylases: 

JMJD1A/KDM2A (+,†) 

JMJD1B/KDM2B (+,†) 

JMJD2A/KDM4A (†,‡) 




KDM4DL/KDM4E (†,‡) 

H3K79me 

Methylases: 

DOTIL/KMT4 (+,†,‡) 

Demethylases: 

 

www.cellsignal.com/cstpathways 27



This table provides 

a referenced list of 

many known histone 

modifications, the 

associated modifying 

enzymes, and proposed 

functions. 

References 

1. Clarke, A.S. et al. (1999) Mol. 

Cell Biol. 19, 2515–2526. 

2. Kimura, A. and Horikoshi, M. 

(1998) Genes Cells 3, 789–800. 

3. Schiltz, R.L. et al. (1999) J. 

Biol. Chem. 274, 1189–1192. 

4. Verreault, A. et al. (1998) 

Curr. Biol. 8, 96–108. 

5. Kawasaki, H. et al. (2000) 

Nature 405, 195–200. 

6. Suka, N. et al. (2001) 

Mol. Cell 8, 473–479. 

7. Angus-Hill, M.L. et al. (1999) 

J. Mol. Biol. 294, 1311–1325. 

8. Sobel, R.E. et al. (1995) 

Proc. Natl. Acad. Sci. USA 

92, 1237–1241. 

9. Grant, P.A. et al. (1999) J. Biol. 

Chem. 274, 5895–5900. 

10. Spencer, T.E. et al. (1997) 

Nature 389, 194–198. 

11. Bird, A.W. et al. (2002) 

Nature 419, 411–415. 

12. Ikura, T. et al. (2000) 

Cell 102, 463–473. 

13. Winkler, G.S. et al. (2002) 


99, 3517–3522. 

14. Hsieh, Y.J. et al. (1999) Mol. 

Cell. Biol. 19, 7697–7704. 

15. Mizzen, C.A. et al. (1996) 

Cell 87, 1261–1270. 

16. Sutton, A. et al. (2003) J. Biol. 

Chem. 278, 16887–16892. 

17. Howe, L. et al. (2001) Genes 

Dev. 15, 3144–3154. 

18. Daujat, S. et al. (2002) 

Curr. Biol. 12, 2090–2097. 

19. Xu, F. et al. (2005) 

Cell 121, 375–385. 

20. Masumoto, H. et al. (2005) 

Nature 436, 294–298. 

21. Parthun, M.R. et al. (1996) 

Cell 87, 85–94. 

22. Kuo, M.H. et al. (1996) 

Nature 383, 269–272. 

23. Kelly, T.J. et al. (2000) Mol. 

Cell. Biol. 20, 7051–7058. 

24. Hilfiker, A. et al. (1997) 

EMBO J. 16, 2054–2060. 

25. Ye, J. et al. (2005) Mol. 

Cell. 18, 123–130. 

26. Briggs, S.D. et al. (2001) 

Genes Dev. 15, 3286–3295. 

27. Wang, H. et al. (2001) 

Mol. Cell. 8, 1207–1217. 

Histone Modifications Table 

The nucleosome, made up of four core histone proteins (H2A, H2B, H3, and H4), and linker histone 

H1 are the primary building blocks of chromatin. Originally thought to function as a static scaffold for 

DNA packaging, histones have more recently been shown to be dynamic proteins, undergoing multiple 

types of post-translational modifications that regulate chromatin condensation and DNA accessibility. 

For example, acetylation of lysine residues has long been associated with histone deposition and 

transcriptional activation, and more recently found to be associated with DNA repair. Phosphorylation 

of serine and threonine residues facilitates chromatin condensation during mitosis and transcriptional 

activation of immediate-early genes. Methylation of lysine and arginine residues function as a major 

determinant for formation of transcriptionally active and inactive regions of chromatin and is crucial for 

proper programming of the genome during development. 

Acetylation 

Histone Site Histone-modifying Enzymes Proposed Function Ref. # 

H2A Lys4 (S. cerevisiae) Esa1 transcriptional activation (1) 

Lys5 (mammals) Tip60, p300/CBP transcriptional activation (2,3) 

Lys7 (S. cerevisiae) Hat1 unknown (4) 

Esa1 transcriptional activation (1) 

H2B Lys5 p300, ATF2 transcriptional activation (3,5) 

Lys11 (S. cerevisiae) Gcn5 transcriptional activation (6) 

Lys12 (mammals) p300/CBP, ATF2 transcriptional activation (3,5) 

Lys15 (mammals) p300/CBP, ATF2 transcriptional activation (3,5) 

Lys16 (S. cerevisiae) Gcn5, Esa1 transcriptional activation (6) 

Lys20 p300 transcriptional activation (3) 

H3 Lys4 (S. cerevisiae) Esa1 transcriptional activation (1) 

Hpa2 unknown (7) 

Lys9 unknown histone deposition (8) 

Gcn5, SRC-1 transcriptional activation (9,10) 


Gcn5, PCAF transcriptional activation (3,11) 

Esa1, Tip60 transcriptional activation (1,2) 

DNA repair (11,12) 

SRC-1 transcriptional activation (10) 

Elp3 transcriptional activation (elongation) (13) 


hTFIIIC90 RNA polymerase III transcription (14) 

TAF1 RNA polymerase II transcription (15) 

Sas2 euchromatin (16) 

Sas3 transcriptional activation (elongation) (17) 

p300 transcriptional activation (3) 

Lys18 Gcn5 transcriptional activation, DNA repair (9) 

p300/CBP DNA replication, transcriptional activation (3,18) 


Gcn5 transcriptional activation, DNA repair (9) 

Sas3 transcriptional activation (elongation) (17) 

p300/CBP transcriptional activation (3,18) 

Lys27 Gcn5 transcriptional activation (6) 


Lys56 (S. cerevisiae) Spt10 transcriptional activation (19) 

DNA repair (20) 

H4 Lys5 Hat1 histone deposition (21) 



ATF2 transcriptional activation (5) 



Acetylation 


Lys8 Gcn5, PCAF transcriptional activation (3,22) 




Elp3 transcriptional activation (elongation) (13) 


Lys12 Hat1 histone deposition (21) 

telomeric silencing (23) 






MOF (D. melanogaster) transcriptional activation (24) 




Sas2 euchromatin (2,6) 

Lys91 (S. cerevisiae) Hat1/Hat2 chromatin assembly (25) 

Methylation 


H1 Lys26 Ezh2 transcriptional silencing (48,49) 

H2A Arg3 PRMT1/6, PRMT5/7 transcriptional activation, transcriptional 

repression 

H3 Arg2 PRMT5, PRMT6 transcriptional repression (83) 

Arg8 PRMT5, PRMT2/6 transcriptional activation, transcriptional (31, 88) 

repression 

Arg17 CARM1 transcriptional activation (18) 



Lys4 Set1 (S. cerevisiae) permissive euchromatin (di-Me) (26) 

Set7/9 (vertebrates) transcriptional activation (tri-Me) (27) 

MLL, ALL-1 transcriptional activation (28,29) 

Ash1 (D. melanogaster) transcriptional activation (30) 

Lys9 Suv39h,Clr4 transcriptional silencing (tri-Me) (32,33) 

G9a 

transcriptional repression genomic 

(34) 

imprinting 

SETDB1 transcriptional repression (tri-Me) (35) 

Dim-5 (N. crassa), 

DNA methylation (tri-Me) (36,37) 

Kryptonite (A. thaliana) 


Lys27 Ezh2 transcriptional silencing (38) 

X inactivation (tri-Me) 

G9a transcriptional silencing (34) 

Lys36 Set2 transcriptional activation (elongation) (39) 

Lys79 Dot1 euchromatin (40) 

transcriptional activation (elongation) (41) 

checkpoint response (42) 

H4 Arg3 PRMT1/6 transcriptional activation (43) 

PRMT5/7 transcriptional repression (31) 

Lys20 PR-Set7 transcriptional silencing (mono-Me) (44) 

Suv4-20h heterochromatin (tri-Me) (45) 


Set9 (S. pombe) checkpoint response (46) 

Lys59 unknown transcriptional silencing (47) 

(83) 

References 

28. Nakamura, T. et al. (2002) 

Mol. Cell. 10, 1119–1128. 

29. Sedkov, Y. et al. (2003) 

Nature 426, 78–83. 

30. Beisel, C. et al. (2002) 

Nature 419, 857–862. 

31. Pal, S. et al. (2004) Mol. 

Cell. Biol. 24, 9630–9645. 

32. Rea, S. et al. (2000) Nature 

406, 593–599. 

33. Nakayama, J. et al. (2001) 

Science 292, 110–113. 

34. Tachibana, M. et al. (2001) 

J. Biol. Chem. 276, 

25309–25317. 

35. Schultz, DzC. et al. (2002) 

Genes Dev. 16, 919–932. 

36. Tamaru, H. and Selker, E.U. 

(2001) Nature 414, 277–283. 

37. Johnson, L. et al. (2002) 

Curr. Biol. 12, 1360–1367. 

38. Cao, R. et al. (2002) 

Science 298, 1039–1043. 

39. Krogan, N.J. et al. (2002) Mol. 

Cell. Biol. 23, 4207–4218. 

40. Feng, Q. et al. (2002) 

Curr. Biol. 12, 1052–1058. 

41. Krogan, N.J. et al. (2003) Mol. 

Cell. 11, 721–729. 

42. Huyen, Y. et al. (2004) Nature 

432, 406–411. 

43. Strahl, B.D. et al. (2001) 

Curr. Biol. 11, 996–1000. 

44. Nishioka, K. et al. (2002) 

Mol. Cell. 9, 1201–1213. 

45. Schotta, G. et al. (2004) 

Genes Dev. 18, 1251–1260. 

46. Sanders, S.L. et al. (2004) 

Cell 119, 603–614. 

47. Zhang, L. et al. (2003) 

Chromosoma 112, 77–86. 

48. Daujat, S. et al. (2005) J. Biol. 

Chem. 280, 38090–38095. 

49. Kuzmichev, A. et al. (2004) 

Mol. Cell 14, 183–193. 

50. Barber, C.M. et al. (2004) 


51. Zhang, Y. et al. (2004) J. Biol. 

Chem. 279, 21866–21872. 

52. Aihara, H. et al. (2004) 

Genes Dev. 18, 877–888. 

53. Harvey, A.C. et al. (2005) 

Genetics 170, 543–553. 

54. Downs, J.A. et al. (2000) 

Nature 408, 1001–1004. 

55. Shroff, R. et al. (2004) Curr. 

Biol. 14, 1703–1711. 

56. Ward, I.M. and Chen, J. 

(2001) J. Biol. Chem. 276, 

47759–47762. 

57. Burma, S. et al. (2001) J. Biol. 

Chem. 276, 42462–42467. 

58. Park, E.J. et al. (2003) Nucleic 

Acids Res. 31, 6819–6827. 

59. Ahn, S.H. et al. (2005) 

Cell 120, 25–36. 

60. Cheung, W.L. et al. (2003) 

Cell 113, 507–517. 


www.cellsignal.com/csttables 29



References 

61. Fernandez-Capetillo, O. et al. 

(2004) J. Exp. Med. 199, 

1671–1677. 

62. Maile, T. et al. (2004) 

Science 304, 1010–1014. 

63. Dai, J. et al. (2004) 

Genes Dev. 19, 472–488. 

64. Hendzel, M.J. et al. (1997) 


65. Hsu, J.Y. et al. (2000) 

Cell 102, 279–291. 

66. Soloaga, A. et al. (2003) 

EMBO J. 22, 2788–2797. 

67. Anest, V. et al. (2003) 

Nature 423, 659–663. 

68. Lo, W.S. et al. (2001) 

Science 293, 1142–1146. 

69. Preuss, U. et al. (2003) Nucleic 

Acids Res. 31, 878–885. 

70. Goto, H. et al. (2002) 

Genes Cells 7, 11–17. 

71. Zhong, S. et al. (2001) J. Biol. 

Chem. 276, 33213–33219. 

72. Cheung, W.L. et al. (2005) 

Curr. Biol. 15, 656–660. 

73. Wang, H. et al. (2004) 

Nature 431, 873–878. 

74. Zhu, B. et al. (2005) 

Mol. Cell. 20, 601–611. 

75. Robzyk, K. et al. (2000) 

Science 287, 501–504. 

76. Nathan, D. et al. (2006) 

Genes Dev. 20, 966–976. 

77. Shiio, Y. and Eisenman, R.N. 

(2003) Proc. Natl. Acad. Sci. 

USA 100, 13225–13230. 

78. Chew, Y.C. et al. (2006) J. 

Nutr. Biochem. 17, 225–233. 

79. Kobza, K. et al. (2005) 

FEBS J. 272, 4249–4259. 

80. Camporeale, G. et al. (2004) 

Eur. J. Biochem. 271, 

2257–2263. 

81. Kothapalli, N. and Zempleni, J. 

(2004) FASEB J. 18, 103–104. 

82. Morris, S.A. et al. (2007) J. 

Biol Chem. 282, 7632–7640. 

83. DiLorenzo, A. et al. (2011) 

FEBS Lett. 585, 2024–2031. 

84. Bungard, D. et al. (2010) 

Science 329, 1201–1205. 

85. Metzger, E. et al. (2010) 

Nature 464, 792–796. 

86. Dawson, M.A. et al. (2009) 

Nature 461, 819–822. 

87. Hurd, P.J. et al. (2009) J. Biol. 

Chem. 284, 16575–16583. 

88. Su, X. et al. (2014) 

Genes Dev. 28, 622–636. 

89. Casadio, F. et al. (2013) 


110, 14894–14899. 

90. Yamagishi, Y. et al. (2010) 

Science 330, 239–243. 

91. Kim, K. et al. (2013) 

Mol. Cell 52, 459–467. 

92. Xiao, A. et al. (2009) 

Nature 457, 57–62. 

Phosphorylation 


H1 Ser27 

transcriptional activation, chromatin 

unknown 

(48,49) 

decondensation 

H2A Ser1 unknown mitosis, chromatin assembly (50) 

MSK1 transcriptional repression (51) 

Ser122 (S. cerevisiae) unknown DNA repair (53) 

Ser129 (S. cerevisiae) Mec1, Tel1 DNA repair (54,55) 

Ser139 (mammalian H2A.X) ATR, ATM, DNA-PK DNA repair (56-58) 

Thr119 (D. melanogaster) NHK1 mitosis (52) 

Thr120 (mammals) Bub1, VprBP mitosis, transcriptional repression (90,91) 

Thr142 (mammalian H2A.X) WSTF apoptosis, DNA repair (92) 

H2B Ser10 (S. cerevisiae) Ste20 apoptosis (59) 

Ser14 (vertebrates) Mst1 apoptosis (60) 

unknown DNA repair (61) 

Ser33 (D. melanogaster) TAF1 transcriptional activation (62) 

Ser36 AMPK transcriptional activation (84) 

H3 Ser10 Aurora-B kinase mitosis, meiosis (64,65) 

MSK1, MSK2 immediate-early gene activation (66) 

IKK-α transcriptional activation (67) 

Snf1 transcriptional activation (68) 

Ser28 (mammals) Aurora-B kinase mitosis (70) 

MSK1, MSK2 immediate-early activation (66,71) 

Thr3 Haspin/Gsg2 mitosis (63) 

Thr6 PKCbI (85) 

Thr11 (mammals) Dlk/Zip mitosis (69) 

Tyr41 JAK2 transcriptional activation (86) 

Tyr45 PKCd apoptosis (87) 

H4 Ser1 unknown mitosis, chromatin assembly (50) 

CK2 DNA repair (72) 

Ubiquitination 


H2A Lys119 (mammals) Ring2 spermatogenesis (73) 

H2B Lys120 (mammals) UbcH6 meiosis (74) 

Lys123 (S. cerevisiae) 

transcriptional activation 

Rad6 

(75) 

euchromatin 

Sumoylation 


H2A Lys126 (S. cerevisiae) Ubc9 transcriptional repression (76) 

H2B Lys6 or Lys7 (S. cerevisiae) Ubc9 transcriptional repression (76) 

H4 N-terminal tail (S. cerevisiae) Ubc9 transcriptional repression (77) 

Biotinylation 


H2A Lys9 biotinidase unknown (78) 

Lys13 biotinidase unknown (78) 

H3 Lys4 biotinidase gene expression (79) 

Lys9 biotinidase gene expression (79) 

Lys18 biotinidase gene expression (79) 

H4 Lys12 biotinidase DNA damage response (80,81) 

Examples of Crosstalk Between Post-Translational Modifications 

Histone H3 

Histone H3 and H4 

p53 

MEF2A 

Transcriptional 

Repression 

Pim-1 

S10 

H3 

14-3-3 

HP1 

S10 

Me 

K9 

H3 

MOF 

K14 

Resting Neurons 

PIAS1 

K403 Su S408 

MEF2A 

K16 

HP1 

dissociation 

P-TEFb 

BRD4 

HATs 

HP1 

DNA Damage 

Aurora B, Ras 

H3 

GCN5 

Membrane Depolarization 

Calcineurin 

Transcription 


Activation 

Chk2 Set7/9 

MDM2 

TIP60 

Ub K372 

K120 

K372 

dissociation 

Me 

S20 

Me K372 

MDM2 

Ub K373 

S20 

CBP/ 

p53 p53 p53 K381 p300 

Ub K381 

Ub K382 

K382 

p53 Ubiquitination/Degradation 

p53 Stability/Transactivation of 

pro-apoptotic target genes 

Dendric Claw 

Differentiation 

Transcriptional Repression 

H4 

S10 

K9 

H3 

K9 

S10 

K403 S408 

MEF2A 

K14 

K14 

RNA Pol II 

No Differentiation 

Transcriptional Activation 

Post-translational modifications (PTMs) are emerging as major effectors of protein function, and in turn, cellular processes. The discovery and investigation of post-translational 

modifications such as methylation, acetylation, phosphorylation, sumoylation, and many others has established both nuclear and non-nuclear roles for PTMs. With the 

awareness of PTMs, there is an ever-growing list of them and more and more research centered on their function. In recent years, there is an overwhelming appreciation for 

the diversity of modifications, but most importantly, the interplay between them. This interplay is essential for proper gene expression, genome organization, cell division and 

DNA damage response. PTMs can directly impact cell function by modifying histones, modifying enzymes and their associated activity, assembling protein complexes as well 

as recognition and targeting in the genome or to other cellular compartments. In the context of single modifications and gene expression, acetylation of certain lysines (i.e., 

Histone 3 lysine [9-H3K9]) correlates with activation, while tri-methylation of this same residue is most often associated with compaction and gene repression. In the case of 

lysine methylation, lysine can be mono-, di-, or tri-methylated; while arginine can be mono- or dimethylated in an asymmetric or symmetric fashion. Each degree of methylation 

for lysines and arginines serves as its own PTM and impacts biological output. Most PTMs do not exist alone in the chromatin environment and the combination of these 

states can reinforce one another. For example, one PTM can serve as a docking site for a binding domain called a “reader” within one protein, while another “reader” within 

the same protein can recognize another residue. This is the case for the reader protein BPTF, which binds both H3K4me3 and H4K16 acetylation. Therefore, modulating the 

various types and degrees of modifications will impact output. For these reasons, the cell has developed a series of enzymes that are important for establishing and maintaining 

these PTMs, which are often referred to as “writers” (e.g., histone methyltransferases, acetyltransferases, etc.) or “erasers” (e.g., histone demethylases, deacetylases, 

etc.). Many of these enzymes have emerged as critical therapeutic targets and have been identified as key regulators of diseases such as cancer. These observations have 

also made their associated PTMs candidates for biomarkers in cancer and other diseases. 


Berger, S.L. (2007) Nature 447, 407–412. • Dawson, M.A., and Kouzarides T. (2012) Cell 150, 12–27. • Gardner, K.E., Allis, C.D., and Strahl, B.D. (2011) J. Mol. Biol. 

409, 36–46. • Lee, J.S., Smith, E., and Shilatifard, A. (2010) Cell 142, 682–685. • Musselman, C.A. and Kutateladze, T.G. (2011) Acids Res. 39, 9061–9071. • Yang, 

X.J. and Seto, E. (2008) Mol. Cell 31, 449–461. 

Ca +2 

© 2006–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Jonathan Whetstine for reviewing this diagram. 




eIF4B is expressed in 

HeLa cells and human 

lung carcinoma. 

Sin1, a component of 

the mTORC2 complex, 

is expressed in many 

cell lines. 

Translational Control 

The synthesis of new proteins is a highly regulated process that allows rapid cellular responses to a 

diverse set of stimuli. Two key events in the control of translational initiation are 1) the association 

between 5’ capped mRNA and the preinitiation complex, and 2) the binding of initiator tRNA to the start 

codon. Both events are mediated by multiple eukaryotic initiator factors (eIFs) that are regulated by 

effector kinases and inhibitors. 

Cap-dependent Initiation 

Translation initiation requires a set of factors to facilitate the association of the 40S ribosomal subunit 

with mRNA. The eIF4F complex, consisting of eIF4E, eIF4A, and eIF4G, binds to the 5ʹ cap structure of 

mRNA. eIF4A is a helicase, and together with accessory protein eIF4B, serves to unwind the secondary 

structure of mRNA at its 5’ untranslated region and promote formation of the preinitiation complex. 

A 

B 

eIF4B (1F5) Mouse mAb #13088: Confocal 

IF analysis of HeLa cells (A) using #13088 

(green). Actin filaments were labeled with 

DyLight 554 Phalloidin #13054 (red). Blue 

pseudocolor = DRAQ5 ® #4084 (fluorescent 

DNA dye). IHC analysis of paraffin-embedded 

human lung carcinoma (B) using #13088. 

Assembly of eIF4F is controlled by growth and survival factors that regulate activity of upstream kinase 

effectors, including Akt, mTOR, p70 S6 kinase, and p90RSK. mTOR kinase complexes mTORC1 and 

mTORC2 promote eIF4F cap-binding complex formation by activating upstream elements that favor 

complex assembly and inhibiting proteins that block eIF4F formation. The mTORC1 complex includes 

mTOR kinase bound by the adaptor raptor and several regulatory proteins (GβL, PRAS40, and DEPTOR), 

while the mTORC2 complex contains mTOR kinase, rictor, GβL, DEPTOR, and Sin1. mTORC1 activates 

p70 S6 kinase to relieve PDCD4 inhibition of eIF4A and activate eIF4B. Initiation factor eIF4B interacts 

with both the eIF3 scaffold protein complex and eIF4A, stimulating eIF4A RNA helicase activity. Upstream 

kinase pathways mediate the phosphorylation of eIF4B by p70 S6 kinase and p90RSK to increase the association 

between eIF4B, eIF3, and eIF4A. Inhibition of translation repressor protein 4E-BP1 by mTORC1 

phosphorylation causes release of cap-binding protein eIF4E and its incorporation into eIF4F. 

Sin1 (D7G1A) Rabbit mAb #12860: WB analysis 

of extracts from various cell lines using #12860. 

Lanes 

1. HeLa 

2. MCF7 

3. Hep G2 

4. INS-1 

5. KNRK 

6. NBT-11 

7. PANC-1 

8. Vero 

9. COS-7 

10. U-87 MG 

11. 293 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

1 2 3 4 5 6 7 8 9 10 11 

Sin1.1 

Sin1.2 


In hypoxic environments, mTORC1 activity is inhibited, leading to down regulation of eIF4E capdependent 

translation. Instead, protein synthesis is driven during low oxygen conditions by eIF4E2 

(also known as 4EHP), which binds the 5ʹ cap and forms a complex with the hypoxia-inducible factor 

HIF-2α and the RNA-binding protein RBM4. This complex stimulates translation of select RNAs, 

including those implicated in cancer growth. 

The 40S ribosomal subunit then binds to the 5ʹ mRNA cap and associated initiation factors and 

searches along the mRNA for the initiation codon. eIF3 physically interacts with eIF4G, which may be 

responsible for the association of the 40S ribosomal subunit with mRNA. 

eIF3H is a core component of the eIF3 complex 

that facilitates binding of mRNA to the 40S ribosomal 

subunit. 

eIF3H (D9C1) XP ® Rabbit mAb #3413: Confocal IF analysis of SK-N-MC cells using 

#3413 (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor 

= DRAQ5 ® #4084 (fluorescent DNA dye). 

Initiator tRNA and the Start Codon 

eIF2 mediates binding of initiator tRNA to the ribosome at the start codon to form the 43S preinitiation 

complex. Trimeric eIF2 is made up of regulatory (α), tRNA/mRNA interacting (β), and GTP/GDP binding 

(γ) proteins. Phosphorylation of eIF2α by multiple upstream kinases (including PKR, PERK, and GCN2) 

is stimulated by environmental stress and the presence of dsDNA, and leads to inactive eIF2 and 

translation inhibition. Additional control of eIF2 activity occurs through regulation of guanine nucleotide 

exchange, which is catalyzed by eIF2B. Exchange of GDP for GTP promotes the essential association 

between the eIF2 complex and tRNA. eIF2B activity is inhibited by GSK-3β phosphorylation and through 

interaction with eIF5, which also acts as a GDP dissociation inhibitor by stabilizing eIF2 bound by GDP. 

Treatment of cells with ER stress-inducing agent 

thapsigargin results in phosphorylation of eIF2α at Ser51. 

Phospho-eIF2α (Ser51) (D9G8) XP ® Rabbit mAb #3398: WB analysis of extracts from C2C12 cells, 

untreated or treated with Thapsigargin #12758, using #3398 (upper) or eIF2α Antibody #9722 (lower). 

eIF2, which transfers Met-tRNA to the 40S subunit 

to form the 43S preinitiation complex, is expressed 

in multiple cell lines. 

eIF2α (D7D3) XP ® Rabbit mAb #5324: WB analysis 


Lanes 

1. MCF7 

2. Hep G2 

3. NIH/3T3 

4. COS-7 

kDa 

140 

100 

80 

60 

50 

40 

30 

1 2 3 4 

elF2α 

kDa 

100 

80 

60 

50 

40 

30 

20 

100 

80 

60 

50 

40 

30 

20 

– + 

PhosphoeIF2α 

(Ser51) 

eIF2α 

Thapsigargin 

S6 ribosomal protein 

is phosphorylated 

by p70 S6 kinase 

at Ser235/236 in 

response to growth 

factors and mitogens. 

Phospho-S6 Ribosomal Protein (Ser235/ 

Ser236) (D57.2.2E) XP ® Rabbit mAb 

#4858: Confocal IF analysis of HeLa cells, 

rapamycin-treated (left) or 20% serum-treated 

(right), using #4858 (green). Actin filaments 

were labeled with Alexa Fluor ® 555 Phalloidin 

#8953 (red). Blue pseudocolor = DRAQ5 ® 

#4084 (fluorescent DNA dye). 

20 


www.cellsignal.com/csttranslational 

33



UV treatment 

results in clustering 

of cytosolic stress 

granules containing 

the translation repressor 

protein TIAR. 

Local Translation 

Some mRNAs are transported in messenger ribonucleoprotein (mRNP) granules to their subcellular 

locations and translated on-site in response to localized signals, known as local translation. This often 

occurs during development, where protein gradients and varying expression patterns are necessary for 

cellular differentiation. mRNPs include stress granules that store mRNA bound to stalled preinitiation 

complexes and the translational repressors TIA-1 and TIAR until translational initiation can begin again 

or the mRNA is degraded. In addition, mRNPs also include cytoplasmic processing bodies (P-bodies) 

that function in mRNA turnover. Together, these elements can control translation, mRNA storage, and 

stability in localized sites. 

EDC4/Ge-1 is an essential component 

of cytoplasmic P-bodies responsible 

for mRNA decapping and degradation. 

EDC4/Ge-1 Antibody #2548: Confocal IF analysis of HeLa cells using 

#2548 (green). Actin filaments were labeled with DY-554 phalloidin (red). 

Blue pseudocolor= DRAQ5 ® #4084 (fluorescent DNA dye). 

Small noncoding RNAs 

Small noncoding RNAs are important regulators of gene expression in higher eukaryotes. Several 

classes of small RNAs, including short interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwiinteracting 

RNAs (piRNAs), have been identified. siRNAs are short segments (20–25 base pairs) of 

double stranded RNA that silence expression of a single gene through complementary base pairing 

that prevents target translation and/or promotes instability. siRNAs are commonly used in the research 

community for antibody validation testing or gene silencing studies. 

Similarly, microRNAs are about 21 nucleotides in length and have been implicated in many cellular 

processes such as development, differentiation, and stress response. miRNAs function together with the 

protein components of complexes called micro-ribonucleoproteins (miRNPs). Among the most important 

components in these complexes are argonaute proteins. Argonaute proteins participate in the various 

steps of microRNA-mediated gene silencing, such as repression of translation and mRNA turnover. 

Silencing of DDX5 expression using DDX5 siRNA. 

SignalSilence ® DDX5 siRNA I #8626 and SignalSilence ® DDX5 siRNA II #8627: WB analysis 

of extracts from HeLa cells, transfected with 100 nM SignalSilence ® Control siRNA (Unconjugated) 

#6568 (-), #8626 (+), or #8627 (+), using DDX5 (D15E10) XP ® Rabbit mAb #9877 (upper) or β-Actin 

(13E5) Rabbit mAb #4970 (lower). The DDX5 (D15E10) XP ® Rabbit mAb confirms silencing of DDX5 

expression, while the β-Actin (13E5) Rabbit mAb is used as a loading control. 

Mili binds to piwi-interacting RNA in male germ 

cells and is essential for spermatogenesis in mouse. 

Mili (D14F5) XP ® Rabbit mAb #5940: Confocal IF analysis of mouse testis using #5940 (green) and 

Pan-Keratin (C11) Mouse mAb #4545 (red). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). 

TIAR (D32D3) XP ® Rabbit mAb #8509: 

Confocal IF analysis of HeLa cells, untreated 

(left) or UV-treated (right), using #8509 


DY-554 phalloidin (red). Blue pseudocolor = 

DRAQ5 ® #4084 (fluorescent DNA dye). 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

60 

50 

– + 

I 

II 

+ 

DDX5 

β-actin 

DDX5 siRNA 


Adjibade, P. and Mazroui, R. (2014) Semin. Cell Dev. Biol. 34, 15–23. • Bar-Peled, L. and Sabatini, D.M. (2014) Trends Cell 

Biol. 24, 400−406. • Donnelly, N., Gorman, A.M., Gupta, S., et al. (2013) Cell Mol. Life Sci. 70, 3493−3511. • Emde, A. 

and Hornstein, E. (2014) EMBO J. 33,1428−1437. • Fabian, M.R., Payette, J., Holcik, M., et al. (2012) Nature 486, 126−129. 

• Hershey, J.W., Sonenberg, N., and Mathews, M.B. (2012) Cold Spring Harb. Perspect. Biol. 4, a011528. • Hinnebusch, A.G. 

and Lorsch, J.R. (2012) Cold Spring Harb. Perspect. Biol. 4, a011544. • Jung, H., Gkogkas, C.G., Sonenberg, N. et al. (2014) 

Cell 157, 26−40. • Kong, J. and Lasko, P. (2012) Nat. Rev. Genet. 13, 383−394. • Spilka, R., Ernst, C., Mehta, A.K., et al. 

(2013) Cancer Lett. 340, 9−21. • Thoreen, C.C. (2013) Biochem. Soc. Trans. 41, 913−916. 

Commonly Studied Translational Control Targets 

Target M P Target M P 

4E-BP1 

• • eIF4A 

• • 

Phospho-4E-BP1 • • eIF4B 

• • PABP1 

(Thr37/Thr46) 

Phospho-eIF4B (Ser406) • • 

Non-phospho-4E-BP1 • Phospho-eIF4B (Ser422) • 

(Thr46) 

eIF4E 

• • PABP2 

Phospho-4E-BP1 (Ser65) • • 

Phospho-eIF4E (Ser209) • PACT 

Phospho-4E-BP1 (Thr70) • • 

eIF4G 

• • Paip2A 

4E-BP2 

• 

Phospho-eIF4G (Ser1108) • PARN 

4EHP 

• 

eIF4GI 

• • PERK 

4E-T 

• 

eIF4G2/p97 • PKR 

ADAR1 

• • 

EIF4H 

• • PPIG 

Argonaute 1 • • 

eIF5 

• 

Argonaute 2 • 

eIF6 

• • PRP4K 


ELAVL1/HuR • PTBP1 


Exportin 5 • • Pumilio 1 

BRF1/2 

• 

FMRP 

• • Pumilio 2 

CLK3 

• 

FUS/TLS 

• RMP 

CNOT2 

• 

FXR1 

• • RPL11 

CNOT3 

• 

FXR2 

• • 

CNOT6 

• 

GCN2 

• 

Coilin 

• 

hnRNP A0 • • 

CPEB1 

• 

hnRNP A1 • • 

DCP1B 

• 

hnRNP C1/C2 • 

DDX3 

• • 

AUF1/hnRNP D • 

DDX4 

• • 

hnRNP E1 

• 

DDX5 

• • 

hnRNP LL 

• 

DDX6/RCK • • 

hnRNP K • • 

DGCR8 

• 

SAM68 

hnRNP Q/R • • 

DHX29 

• • 

SF2/ASF 

IMP1 

• • 

Dicer1 

• • 

SF3B1 

IWS1 

• 

Drosha 

• 

SKAR 

KHSRP 

• • 

EDC4/Ge-1 

• 

SKAR α/β 

La Antigen • • 

eEF1A 

• • 

SMN1 

LSm2 

• 

eEF2 

• 

Symplekin 

LysRS 

• • 

Phospho-eEF2 (Thr56) • 

TFEB 

MAPBPIP/ROBLD3/p14 • 

eEF2k 

• 

THEX1 

MAPKSP1/MP1 • 

Phospho-eEF2k (Ser366) • 

MetAP2 • 

eIF1 

• 

TIAR 

Mili 

• • 

eIF2α 

• • 

U2AF1 

Miwi 

• • 

Phospho-eIF2α (Ser51) • • 

Upf1 

Mnk1 

• 

eIF2B-ε 

• 

Upf2 

Phospho-Mnk1 • 

eIF3A 

• • 

XBP-1s 

(Thr197/202) 

eIF3C 

• 

XRN2 

MRPL11 • • 

eIF3H 

• 

ZPR1 

NCBP1/CBP80 • 

eIF3J 

• • NRF1/TCF11 • 

eIF4A1 

• NRF2 

• 

Target M P 

NXF1 

Asymmetric-Methyl-PABP1 

(Arg455/Arg460) 

Phospho-PPIG (Ser376) 

S6 Ribosomal Protein 

Phospho-S6 Ribosomal 

Protein (Ser235/Ser236) 

Phospho-S6 Ribosomal 

Protein (Ser240/Ser244) 

Ribosomal Protein L7a 

Ribosomal Protein L13a 

Ribosomal Protein L26 

Ribosomal Protein S3 

THOC4/ALY 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• • 

• • 

• 

• 

• • 

• • 

• 

• 

• 

• 

• • 

• 

• 

• 

• • 

• 

• • 

• 

• • 

• 

• 

• 

• 


nodes within translational control 

signaling pathways and are commonly 

studied in translational control research. 

Primary antibodies, antibody conjugates, 

and antibody sampler kits containing 

these targets are available from CST. 





207 


CST antibodies for Phospho-S6 

Ribosomal Protein (Ser235/236) 

have been cited over 207 times in 

high-impact, peer-reviewed publications 



Wong, C.C. et al. (2014) Inactivating 

CUX1 mutations promote tumorigenesis. 

Nat. Genet. 46, 33−38. 

Kurachi, M. et al. (2014) The 

transcription factor BATF operates as 

an essential differentiation checkpoint 

in early effector CD8+ T cells. Nat. 

Immunol. 15, 373−383. 

Mouw, J.K. et al. (2014) Tissue 

mechanics modulate microRNAdependent 

PTEN expression to 

regulate malignant progression. 

Nat. Med. 20, 360−367. 

Agarwal, A. et al. (2014) Antagonism 

of SET using OP449 enhances the 

efficacy of tyrosine kinase inhibitors 

and overcomes drug resistance in 

myeloid leukemia. Clin. Cancer Res. 

20, 2092−2103. 

Koo, J. et al. (2014) Maintaining 

glycogen synthase kinase-3 activity 

is critical for mTOR kinase inhibitors 

to inhibit cancer cell growth. Cancer 

Res. 7, 2555−2568. 

Fay, M.M. et al. (2014) Enhanced 

Arginine Methylation of Programmed 

Cell Death 4 Protein during Nutrient 

Deprivation Promotes Tumor 

Cell Viability. J. Biol. Chem. 289, 

17541−17552. 


www.cellsignal.com/csttranslational 

35


Translational Control: Overview 

eEF2K 

GTP 

eEF1 

Cap 

Cap 

eEF2 

GTP 

AUG 

eEF2 Control: 

Ca 2+ 

cAMP 

mTORC1 

40S 

AUG 

60S 

eIF5B 

GDP 

Elongation 

40S 

60S 

Factor 

Release 

eIF3 

A (n) 

eIF1A 

Nascent 

Polypeptide 

ER Lumen 

Initiation Codon 

Recognition 

eIF2 

GDP 

A(n) 

eIF5 

Cap 

eIF1 

Polypeptide 

Chain 

eIF4 Control: 

Growth Factors (mTORC1), 

Hormones, Cytokines, Mitogens, 

Neuropeptides, Oxidative Stress 

48S 

AUG 

Cap 

eIF6 

eIF5B 

GTP 

60S 

A (n) 

Initiation 

Stress Granules 

P-bodies 

40S 

Scanning 

AAAAA 

DHX29 

miRNA 

60S 

48S 

Cap 

AUG 

eIF4B 

eRF1 

eRF3 

GTP 

A (n) 

ON 

eIF4F 

Cap 

PABP AUG 

A (n) 

Termination 

eIF2 Control: 

Viral Infection (dsRNA), (PKR) 

Amino Acid Starvation, UV Light (GCN2) 

Heme Deficiency (HRI) 

Heat Shock, ER Stress, Hypoxia (PERK) 

miRNA 

43S 

eIF2 

Met-tRNAi 

GTP 

eIF3 

40S 

Initiation 

Complex 

The synthesis of new proteins is a highly regulated process that allows rapid cellular responses to diverse stimuli at the post-transcriptional level. Nine key eukaryotic translation 

initiation factors (eIFs) catalyze the assembly of a functional ribosomal complex in two steps - first, the formation of the 48S complex from the 43S initiation complex 

and mRNA followed by its subsequent joining with the 60S subunit, enabling polypeptide chain formation. Of the many steps in translation, the rate-limiting step, initiation, is 

subjected to the most regulatory control. Many stimuli, such as growth factors and stress, either stimulate or inhibit specific eIFs. Aside from initiation, translation can also be 

attenuated during elongation. For instance, elevated levels of Ca 2+ or cAMP can block the action of eukaryotic elongation factor 2 (eEF2) via AMPK. Finally, upon recognition of 

a stop codon, eRF1 and eRF3 mediate termination of translation and ribosome disassembly and recycling. 


Dever, T.E. and Green, R. (2012) Cold Spring Harb. Perspect. Biol. 4, a013706. • Gebauer, F., and Hentze, M.W. (2004) Nat. Rev. Mol. Cell Biol. 5, 827–835. • Hinnebusch, 

A.G. (2011) Microbiol. Mol. Biol. Rev. 75, 434–467. • Sonenberg, N. and Hinnebusch, A.G. (2009) Cell 136, 731–745. • Spirin, A.S. (2009) Biochemistry 48, 10688– 

10692. • Steitz, T.A. (2008) Nat. Rev. Mol. Cell Biol. 9, 242–253. 

eIF1A 

eIF1 

eIF5 

OFF 

Translational Control: Regulation of elF4E and p70 S6K 

Amino 

Acids 

GRB10 

Hormones, Growth Factors, 

Cytokines, Neuropeptides Mitogens Stress 

IRS-1 

PI3K 

RagA/B 

RagC/D 

4E- 

BP1 eIF4E 

Translation Off 

mTORC1 

GβL Raptor 

mTOR 

DEPTOR 

AMP: 

ATP 

LKB1 

AMPK 

PIP 3 

PDK1 

Akt 

TSC2 

TSC1 TBC1D7 

PRAS40 rapamycin 

FKBP12 

mTORC1 

mTORC2 

Sin1 PRR5 

Rictor GβL 

mTOR 

DEPTOR 

S6 

4E- 

BP1 


PTEN 

mTORC2 

GSK-3 

Torin1 

PP242 

KU63794 

WYE354 

p70 S6K 

PDCD4 

eIF4B 

elF4A eIF4H 

MNK 

eIF4E eIF4G 

eIF4F 

Cap 

PABP 

AAAAA 

eIF4F 

LRP 

Wnt 

Frizzled 

Gα q/o 

Dvl 

Erk 

p90RSK 

PABP 

PAIP1 

PAIP2 

43S 

48S 

p38 MAPK 

eEF2K eEF2 

Translation Elongation On 

Translation On 

Translation is a tightly regulated process, and the mTORC1-S6K signaling axis plays a critical role in this control. The rate of translation initiation is predominantly determined 

by 5’ cap recognition by eIF4F, a trimeric protein complex composed of eIF4E, which binds the 5ʹ cap; eIF4A, a helicase necessary for unwinding complex secondary structure 

in the leader sequence; and eIF4G, a large scaffolding protein that delivers the mRNA to eIF3 and mediates mRNA circularization through association with polyA binding 

protein (PABP). Binding of eIF4F to the cap is hindered by eIF4E binding proteins (4EBPs), which, when hypophosphorylated, sequester eIF4E and prevent its association 

with eIF4G. However, in response to positive stimuli such as growth factors, mitogens, and amino acids, mTORC1 phosphorylates 4EBPs and relieves this inhibition, allowing 

the formation of eIF4F and subsequent initiation of translation. In addition, mTORC1 - alongside PDK1 - phosphorylates S6 kinase, which in turn phosphorylates numerous 

substrates involved in translation. These include S6 small ribosomal subunit; eIF4B, an activator of the eIF4A helicase; PDCD4, an eIF4A inhibitor that is inhibited by phosphorylation; 

and SKAR, an mRNA splicing factor. Aside from the mTORC1 pathway, the Ras-MAPK pathway is another major regulator of translation and is responsible for the 

phosphorylation of eIF4B as well as eIF4E, via MNK kinases. 


Dowling, R.J., Topisirovic, I., Fonseca, B.D., and Sonenberg, N. (2010) Biophys. Acta. 1804, 433–439. • Fenton T.R. and Gout, I.T. (2011) Int. J. Biochem. Cell Biol. 43, 

47–59. • Graff, J.R., Konicek, B.W., Carter, J.H., and Marcusson, E.G. (2008) Cancer Res. 68, 631–634. • Holcik, M. and Sonenberg, N. (2005) Nat. Rev. Mol. Cell Biol. 6, 

318–327. • Huang, J. and Manning, B.D. (2008) Biochem. J. 412, 179–190. • Magnuson, B., Ekim, B., and Fingar, D.C. (2012) Biochem. J. 441, 1–21. • Ruvinsky, I. 

and Meyuhas, O. (2006) Trends Biochem. Sci. 31, 342–348. • Sonenberg, N. and Hinnebusch, A.G. (2009) Cell 136, 731–745. 

© 2002–2015 Cell Signaling Technology, Inc. • We would like to thank Rachel Wolfson and Prof. David Sabatini, Whitehead Institute for Biomedical Research, MIT, Cambridge, MA, for reviewing this diagram. 



www.cellsignal.com/cstpathways 

37



Translational Control: Regulation of elF2 

PTEN 

Salubrinal 

ATF-4, CHOP, mRNA 

Translation 

α 

GDP 

Apoptosis, Changes in 

Metabolism & Redox Status 

SirT1 

eIF2B 

eIF2 γ 

Akt 

Growth Factors, 

ER Stress, 

Viral Infection 

dsRNA 

PKR 

GADD34 

eIF2B 

β 

Unfolded Protein 

Response, Hypoxia 

PKR 

PP1 

CreP 

eIF5 

PERK 

PERK 

PP1 

SirT1 

Global Translation Off 

40S 

eIF3 

eIF5 

BiP 

α 

GDP 

Glucose Deprivation, 

UV Light, Amino Acid 

Starvation 

α 

GDP 

GDP 

α 

GTP 

GCN2 

GCN2 

eIF2 γ 

eIF2 γ 

eIF2 γ 

β 

β 

β 

GTP 

eIF2 

Met-tRNAi 

Ternary 

Complex 

43S 

GTP 

Global Translation On 

eIF5 

HRI 

eIF5 

Met-tRNAi 

eIF1 

eIF1A 

Heme Deficiency, 

Oxidative Stress 

HRI 

NCK 

eIF2B 

GSK-3β 


Hormones, etc. 

The eIF2 initiation complex integrates a diverse array of stress-related signals to regulate both global and specific mRNA translation. Under permissive conditions, eIF2 binds 

GTP and Met-tRNAi to form the ternary complex (TC), which then associates with the 40S ribosomal subunit, eIF1, eIF1A, eIF5, and eIF3 to form the 43S pre-initiation complex 

(PIC). The 43S PIC scans the mRNA UTR for an AUG start codon. Upon AUG recognition, eIF2 hydrolyzes GTP to GDP with the help of the GTPase activating protein eIF5 

and dissociates from the mRNA, permitting the binding of the 60S ribosomal subunit and elongation of the polypeptide chain. eIF2 remains bound to GDP in the presence of 

eIF5 acting as a GDI. To permit another round of initiation, eIF2B must act as both a GDI displacement factor (GDF) and a guanine exchange factor (GEF) to allow exchange of 

GDP for GTP on eIF2. This step is tightly regulated, and phosphorylation of eIF2α by a diverse family of four stress activated kinases—PKR (dsRNA), PERK (ER stress), GCN2 

(amino acid starvation), and HRI (heme deficiency)—prevents nucleotide exchange by causing eIF2 to act as a dominant negative complex to sequester eIF2B. The resulting 

increase in eIF2α-GDP limits the availability of the ternary complex and causes a decrease in global protein synthesis and an enhancement of the translation of specific stressrelated 

mRNA transcripts, such as the transcription factors ATF-4 and CHOP. 


Hinnebusch, A.G. (2011) Microbiol. Mol. Biol. Rev. 75, 434–467. • Raven, J.F. and Koromilas, A.E. (2008) Cell Cycle 7, 1146–1150. • Schmitt, E., Naveau, M., and 

Mechulam, Y. (2010) FEBS Lett. 584, 405–412. • Stolboushkina, E.A. and Garber, M.B. (2011) Biochemistry 76, 283–294. • Wek, R.C., Jiang, H.Y., and Anthony, T.G. 

(2006) Biochem. Soc. Trans. 34, 7–11. 

Nuclear Receptors 

The nuclear receptor superfamily are ligand-activated transcription factors that play diverse roles in cell 

differentiation/development, proliferation, and metabolism and are associated with numerous pathologies 

such as cancer, cardiovascular disease, inflammation, and reproductive abnormalities. Members 

of this family contain an N-terminal transactivation domain, a highly conserved central region zinc-finger 

DNA binding domain, and a C-terminal ligand-binding domain. Ligand binding to its correlate nuclear 

receptor results in transactivation of specific genes within a target tissue. 

In addition to ligand binding, nuclear receptor activity can be modulated through the action of numerous 

growth factor and cytokine signaling cascades that result in receptor phosphorylation or other 

post-translational modifications, typically within the N-terminal transactivation domain. For example, 

the estrogen receptor is phosphorylated on multiple serine residues that affect receptor activity. Ser118 

may be the substrate of the transcription regulatory kinase CDK7, whereas Ser167 may be phosphorylated 

by p90RSK and Akt. Phosphorylation of Ser167 may confer resistance to tamoxifen in breast 

cancer patients. 

Type I Nuclear Receptors 

Type I nuclear receptors, also called steroid receptors, include the estrogen receptor, androgen 

receptor, progesterone receptor, mineralocorticoid receptor, and glucocorticoid receptor. Steroid 

hormone ligands for this subgroup of receptors travel from their respective endocrine gland through 

the bloodstream bound to steroid binding globulin. Some type I nuclear receptors are activated, in part, 

upon binding their respective ligand in the cytoplasmic compartment. The ligand-receptor complex dissociates 

from HSP90 and enters the nucleus where it homodimerizes and binds to hormone response 

elements within the promoter of a target gene. The receptor transactivation domain is responsible for 

interaction at the promoter with co-activators such as acetyltransferases and the general transcription 

machinery, resulting in transcriptional activation. 

Androgen receptor, a type I nuclear receptor, plays a crucial role in several 

stages of male development and the progression of prostate cancer. 

A 

Androgen Receptor (D6F11) XP ® Rabbit mAb #5153: IHC analysis of paraffin-embedded human prostate carcinoma (A) using #5153. 

Confocal IF analysis of LNCaP (positive) (B) and DU 145 (negative) (C) cells using #5153 (green). Actin filaments have been labeled with 

DY-554 phalloidin (red). 

Dexamethasone treatment results in translocation of the glucocorticoid 

receptor to the nucleus, where it associates with response elements within 

glucocorticoid-responsive genes. 

A 

B 

B 

C 

C 

Commonly Studied 

Nuclear Receptor 

Targets 


nodes within nuclear receptor signaling 

pathways and are commonly studied 

in nuclear receptor research. Primary 








Target M P 

AhR 

• 

Androgen Receptor • • 

Aromatase 

• 

COUP-TF1 • 

COUP-TF2 • 

Estrogen Receptor-α • 

Phospho-Estrogen • 

Receptor-α (Ser104/Ser106) 


Receptor-α (Ser118) 


Receptor-α (Ser167) 

ERRα 

• 

Glucocorticoid Receptor • 

Phospho-Glucocorticoid • 

Receptor (Ser211) 

NRBF-2 

• • 

Nur77 

• 

Phospho-Nur77 (Ser351) • 

PHB2 

• • 

PPARγ 

• • 

Progesterone Receptor • 

Phospho-Progesterone • 






Progesterone Receptor A/B • • 

Progesterone Receptor B • • 

RARα 

• 

RARγ 

• 

Rev-Erba • 

Phospho-Rev-erba • 

(Ser55/59) 

RXR-α 

• • 

RXRβ 

• 

RXRγ 

• 

STF-1 

• 

Vitamin D3 Receptor • 



Glucocorticoid Receptor (D6H2L) XP ® Rabbit mAb #12041: IHC analysis of paraffin-embedded human prostate carcinoma (A) using 

#12041. Confocal IF analysis of HeLa cells, grown in phenol red-free media containing 5% charcoal-stripped FBS for 2 d and either untreated 

(B) or treated with dexamethasone (100 nM, 2 hr) (C), using #12041 (green). Actin filaments were labeled with DY-554 phalloidin (red). 

www.cellsignal.com/cstnuclear 39


8 


CST antibodies for Androgen Receptor 





Cuenca-Lopez, M.D. et al. (2014) 

Phospho-kinase profile of triple 

negative breast cancer and androgen 

receptor signaling. BMC Cancer 

14, 302. 

Boll, K. et al. (2013) MiR-130a, 

miR-203 and miR-205 jointly repress 

key oncogenic pathways and are 

downregulated in prostate carcinoma. 

Oncogene 32, 277−285. 

Krycer, J.R. et al. (2013) Does changing 

androgen receptor status during 

prostate cancer development impact 

upon cholesterol homeostasis PLoS 

One 8, e54007. 

Nie, H. et al. (2013) Acetylcholine 

acts on androgen receptor to promote 

the migration and invasion but inhibit 

the apoptosis of human hepatocarcinoma. 

PLoS One 8, e61678. 

Furu, K. et al. (2013) Tzfp represses 

the androgen receptor in mouse 

testis. PLoS One 8, e62314. 

Li, Y. et al. (2013) Functional domains 

of androgen receptor coactivator p44/ 

Mep50/WDR77and its interaction 

with Smad1. PLoS One 8, e64663. 

Ota, H. et al. (2012) Testosterone 

deficiency accelerates neuronal 

and vascular aging of SAMP8 mice: 

protective role of eNOS and SIRT1. 


Krycer, J.R. et al. (2011) Cross-talk 

between the androgen receptor and 

the liver X receptor: implications for 

cholesterol homeostasis. J. Biol. 

Chem. 286, 20637−20647. 

Type II Nuclear Receptors 

Type II nonsteroid nuclear receptors include the thyroid hormone receptors (TRα and β), retinoic acid 

receptors (RARα, β, and γ), vitamin D receptor (VDR), and peroxisome proliferator-activated receptors 

(PPARα, β, and γ). Members of this family heterodimerize with the retinoid X receptor (RXR). Prior to 

ligand binding, receptor heterodimers are located in the nucleus as part of complexes with histone 

deacetylases (HDACs) and other co-repressors that keep target DNA in a tightly wound conformation, 

preventing exposure to transacting factors. Ligand binding results in co-repressor dissociation, 

chromatin derepression, and transcriptional activation. 

RARγ1, a type II nuclear receptor that regulates expression 

of genes involved in cellular differentiation, proliferation, and 

apoptosis, is expressed in cancer cells and epidermal cells. 

RARγ1 (D3A4) XP ® Rabbit mAb #8965: 

WB analysis of extracts from various cell lines 

(A) using #8965. IHC analysis of paraffinembedded 

human skin (B) using #8965. 

Lanes 

1. HaCaT 

2. A-375 

3. BxPC-3 

4. T-47D 

5. SK-BR-3 

A 

kDa 

100 

80 

60 

50 

40 

1 2 3 4 5 

Orphan Nuclear Receptors 

Orphan nuclear receptors are nuclear receptors where the endogenous ligands have not been identified. 

Structural studies suggest that some of the orphan receptors may not bind ligands. This class of nuclear 

receptors includes small heterodimer partner (SHP), reverse orientation c-ErbA (Rev-Erbα and β), 

testicular receptor 2 and 4 (TR2 and 4), tailless homolog orphan receptor (TLX), photoreceptor-specific 

NR (PNR), chicken ovalbumin upstream promoter transcription factor 1 and 2 (COUP-TF1 and 2), Nur77, 

Nur-related protein 1 (NURR1), neuron derived orphan receptor 1 (NOR1), estrogen-related receptor 

(ERR α, β, and γ), and germ cell nuclear factor (GCNF). Most of these receptors regulate transcription 

by binding to their target DNA elements either as monomers or homodimers and recruiting chromatin 

modifying coactivators and the transcription machinery. Nur77 and NURR1 can also heterodimerize 

with RXRs and these heterodimers are able to respond to RXR ligands to regulate transcription. 

Rev-Erbα (E1Y6D) Rabbit mAb #13418: Chromatin IPs were performed 

with cross-linked chromatin from 4 x 10 6 Hep G2 cells and either 10 μl of 

#13418 or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP ® Enzymatic 

Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified 

by real-time PCR using human BMAL1 promoter primers, SimpleChIP ® Human 

NR1D1 Promoter Primers #13413, and SimpleChIP ® Human α Satellite 

Repeat Primers #4486. The amount of immunoprecipitated DNA in each 

sample is represented as a percent of the total input chromatin. 

Rev-Erbα (E1Y6D) Rabbit 

mAb #13418 

Normal Rabbit 

IgG #2729 


RARγ1 

Rev-Erbα, an orphan nuclear receptor involved in 

cell proliferation, differentiation, and circadian rhythms. 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

BMAL1 

NR1D1 

B 

α Satellite 



Ahmadian, M., Suh, J.M., and Hah, N. et al. (2013) Nat. Med. 19, 557−566. • Bondesson, M., Hao, R., Lin, C.Y., Williams, C., and Gustafsson, J.A. (2014) Biochim. 

Biophys. Acta. Jun 17 [Epub ahead of print]. • Evans, R.M. and Mangelsdorf, D.J. (2014) Cell 157, 255−266. • Kojetin, D.J. and Burris, T.P. (2014) Nat. Rev. Drug Discov. 

13, 197−216. • Kurakula, K., Koenis, D.S., van Tiel, C.M., and de Vries, C.J. (2014) Biochim. Biophys. Acta. 1843, 2543–2555. • Manolagas, S.C., O’Brien, C.A., and 

Almeida, M. (2013) Nat. Rev. Endocrinol. 9, 699−712. • Zhou, W. and Slingerland, J.M. (2014) Nat. Rev. Cancer 14, 26−38. 

Nuclear Receptors Signaling 

HSP90 

Type I Steroid Receptors (Homodimers) 

NR 

NR 

NR 

NR 

HRE 

SBP 

HSP90 

Homodimerization 

SRC1/2 PRMT/ 

CARM 

CBP/ 

p300 PCAF 

RNA 

TBP TFIIB POLII 

TATA 

Plasma Membrane 

Cytoplasm 

Nucleus 


Activation 

Type IIa Non-steroid Receptors (RXR Heterodimers) 

Heterodimerization 

NcoR1 

SMRT 

RXR 

NR 

Ligand 

Plasma Membrane 

Cytoplasm 

Nucleus 

Type IIb Orphan Receptors (Monomers and Homodimers) 

NcoR1 

SMRT 

NR 

NR 

RXR 

NR 

HRE 

HDAC 

Complex 

No Ligand 

Transcriptional Repression 

NR 

NR 

HRE 

HDAC 

Complex 

SRC1/2 

PRMT/ 

CARM 

CBP/ 

p300 PCAF 

RNA 


SRC1/2 

PRMT/ 

CARM 

CBP/ 

p300 PCAF 

RNA 


Constitutive 


Repression 


Activation 

Nucleus 

Constitutive 


Activation 

Nur77, an orphan 

nuclear receptor 

involved in cell 

proliferation, 

differentiation, 

and apoptosis 

Nur77 (D63C5) XP ® Rabbit mAb #3960: 

Confocal IF analysis of Jurkat cells, untreated 

(left) or treated with TPA #4174 and A23187 

(right), using #3960 (green). Actin filaments 

were labeled with DY-554 phalloidin (red). 

© 2012–2015 Cell Signaling Technology, Inc. 



02 


Activation of MAPK 

signaling with TPA 

results in phosphorylation 

of Erk1/2 at 

Thr202/Tyr204. 

Signaling 

MAP Kinase Signaling 

Mitogen-activated protein kinases (MAPKs) are a highly conserved family of serine/threonine protein 

kinases involved in a variety of fundamental cellular processes such as proliferation, differentiation, 

motility, stress response, apoptosis, and survival. Conventional MAPKs include the extracellular 

signal-regulated kinase 1 and 2 (Erk1/2 or p44/42), the c-Jun N-terminal kinases 1-3 (JNK1-3)/ 

stress activated protein kinases (SAPK1A, 1B, 1C), the p38 isoforms (p38α, β, γ, and δ), and Erk5. The 

lesser-studied, atypical MAPKs include Nemo-like kinase (NLK), Erk3/4, and Erk7/8. 

A 

B 

C 

kDa 

80 

60 

50 

40 

30 

80 

60 

50 

40 

30 

293 

NIH/3T3 

C6 

Phosphop44/42 

MAPK 

(Thr202/Tyr204) 

+ – + – + – 

p44/42 

MAPK 

λ phosphatase 

– + – + – + TPA 

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP ® Rabbit mAb #4370: Confocal IF analysis of C2C12 cells, treated 

with U0126 #9903 (10 μM for 1 hr) (A) or TPA #9905 (200 nM for 15 min) (B), using #4370 (green). Actin filaments were labeled with 

Alexa Fluor ® 555 Phalloidin #8953 (red). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). WB analysis of extracts from 293, 

NIH/3T3, and C6 cells, treated with λ phosphatase or TPA #4174 as indicated (C), using #4370 (upper), or p44/42 MAPK (Erk1/2) (137F5) 

Rabbit mAb #4695 (lower). 

Signaling via the conventional MAPKs follows a classical 

three-tiered kinase cascade: MAPKKK MAPKK MAPK. 

CONVENTIONAL MAPKS 

ATYPICAL MAPKS 

A broad range of extracellular stimuli including mitogens, cytokines, growth factors, and environmental 

stressors stimulate the activation of one or more MAPKK kinases (MAPKKKs) via receptor-dependent 

and -independent mechanisms. MAPKKKs then phosphorylate and activate a downstream MAPK 

kinase (MAPKK), which in turn phosphorylates and activates MAPKs. Activation of MAPKs leads to the 

phosphorylation and activation of specific MAPK-activated protein kinases (MAPKAPKs), such as members 

of the RSK, MSK, or MNK family, and MK2/3/5. These MAPKAPKs function to amplify the signal 

and mediate the broad range of biological processes regulated by the different MAPKs. While most 

MAPKKK, MAPKK, and MAPKs display a strong preference for one set of substrates, there is significant 

cross-talk in a stimulus- and cell-type–dependent manner. 

Activation of MAPK signaling by TPA results in 

phosphorylation of c-Fos, a nuclear oncogene 

that dimerizes with c-Jun to form the AP-1 

transcription factor. 

Phospho-c-Fos (Ser32) (D82C12) XP ® Rabbit mAb (PE Conjugate) #11919: 

Flow cytometric analysis of HeLa cells, untreated (blue) or treated with TPA #4174 

(green), using #11919. 

Events 

Phospho-c-Fos (Ser32) (PE Conjugate) 

UV treatment activates the p38 MAPK signaling pathway, resulting 

in phosphorylation of MKK3 at Ser189 and MKK6 at Ser207. 

Phospho-MKK3 (Ser189)/MKK6 

(Ser207) (D8E9) Rabbit mAb #12280: 

Confocal IF analysis of HeLa cells, 

untreated (left) or UV-treated (40 mJ/ 

cm 2 with 30 min recovery; right), using 

#12280 (green). Actin filaments were 

labeled with DY-554 phalloidin (red). 

Blue pseudocolor = DRAQ5 ® #4084 

(fluorescent DNA dye). 

chapter 02: Signaling 

Activation of MAPK 

signaling with TPA 


of MEK1/2 at 

Ser217/221. 

kDa 

140 

100 

80 

60 

50 

40 

30 

100 

80 

60 

50 

40 

HeLa 

NIH/3T3 

Phospho- 

MEK1/2 

(Ser217/221) 

MEK1/2 

+ – + – TPA 

Phospho-MEK1/2 (Ser217/221) 

(41G9) Rabbit mAb #9154: WB 

analysis of extracts from untreated or 

TPA-treated HeLa and NIH/3T3 cells 

using #9154 (upper) or MEK1/2 Antibody 

#9122 (lower). 

Phopho-p38 MAPK 

(Thr180/Tyr182) is 

expressed in human 

colon carcinoma. 

Stimulus 


Mitogens, GPCR, 

Antigen Receptors 

Stress, DNA Damage, GPCR, 

Inflammatory Cytokines, 

Growth Factors 

Stress, Mitogens, GPCR, 

Neurotrophic Factors, 

GPCR, RTKs, 

Neurotrophic Factors 

Activator 

MAPKKK 

MAPKK 

RAS 

A-Raf, 

B-Raf, c-Raf, 

Mos, Tpl2 

MEK1/2 

Ras, Rho, Rac, 

Cdc42, TRAFs, 

GADD45a 

MEKK3/4, 

ASK1/2, TAOK1/2, 

MLK3, Tpl2, 

DLK, ZAK 

MKK3/6, 

MKK4 

Ras, Rho, Rac, 

Cdc42, TRAFs, 

GADD45a 

MEKK1/2/4, 

MLK1-4, ASK1/2, 

TAOK1/2,TAK1, 

DLK, ZAK 

Gαq, Gα12/13, 

Ras, Rap 

MEKK2/3 

MKK4/7 MEK5 PAK1/2/3 

Unknown 

Anisomycin treatment results in activation and nuclear 

translocation of phospho-SAPK/JNK (Thr183/Tyr185). 

Phospho-SAPK/JNK (Thr183/Tyr185) 

(G9) Mouse mAb #9255: Confocal IF 

analysis of HeLa cells, untreated (left) 

and anisomycin-treated (right), using 



Phospho-p38 MAPK (Thr180/Tyr182) 

(D3F9) XP ® Rabbit mAb #4511: IHC 

analysis of paraffin-embedded human 

colon carcinoma using #4511. 

MAPK 

Erk1/2 

p38-MAPK 

α/β/γ/δ 

JNK1-3 Erk5 Erk3/4 Erk7/8 

MAPKAPK 

Biological 

Response 

RSK1-4, 

MNK1/2, 

MSK1/2 

Growth, Differentiation, 

Proliferation, Development 

MAPKAPK-2/3, 

MSK1/2, MNK1 

MAPKAPK-2, 

MAPKAPK-3 

Inflammation, Apoptosis, 

Growth, Differentiation 

RSK1-4 

Growth, 

Differentiation, Development 

MAPKAPK-5 

Inflammation, Apoptosis, 



Arthur, J.S. and Ley, S.C. (2013) Nat. Rev. Immunol. 13, 679–692. • Cargnello, M. and Roux, P.P. (2011) Microbiol. Mol. Biol. 

Rev. 75, 50–83. • Cseh, B., Doma, E., and Baccarini, M. (2014) FEBS Lett. 588, 2398–2406. • Darling, N.J. and Cook, S.J. 

(2014) Biochim. Biophys. Acta. 1843, 2150–2163. • Koul, H.K., Pal, M., and Koul, S. (2013) Genes Cancer 4, 342–359. • 

Plotnikov, A., Zehorai, E., Procaccia, S., and Seger, R. (2011) Biochim. Biophys. Acta. 1813, 1619–1633. • Sehgal, V. and 

Ram, P.T. (2013) Genes Cancer 4, 409–413. 


www.cellsignal.com/cstmapk 43




nodes within MAP Kinase pathways 

and are commonly studied in MAP 

Kinase research. Primary antibodies, 

antibody conjugates, and antibody 

sampler kits containing these targets 

are available from CST. 








Chemical Modulators for the Study of MAP Kinase Signaling 

#2222 Anisomycin Inhibits protein synthesis; activates stress-activated kinases (SAPK/JNK, p38-MAPK) 

#11916 Chelerythrine 

Chloride 

SP600125 is a highly specific inhibitor of JNK-family kinases. 

SP600125 #8177: WB analysis of extracts from 293T cells, untreated or 

treated with Anisomycin #2222 (25 μg/ml) for the indicated times either with 

or without #8177 pre-treatment (50 μM, 40 min), using Phospho-JunB 

(Thr102/Thr104) (D3C6) Rabbit mAb #8053 (upper) or JunB (C37F9) Rabbit 

mAb #3753 (lower). 

Commonly Studied MAP Kinase Targets 


ASK1 

• • 

Phospho-ASK1 (Ser83) • 

Phospho-ASK1 (Thr845) • 

Phospho-ASK1 (Ser967) • 

APS 

• 

β-Arrestin 1 • • 

Phospho-β-Arrestin 1 (Ser412) • 

β-Arrestin 1/2 

• 

β-Arrestin 2 

• 

ATF-2 • • 

Phospho-ATF-2 (Thr69/Thr71) • 

Phospho-ATF-2 (Thr71) • • • • 

Bcr 

• 

Phospho-Bcr (Tyr177) 

• 

Bcr-Abl (b2a2 Junction Specific) • 

c-Abl 

• 

Phospho-c-Abl (Tyr89) • 

Phospho-c-Abl (Tyr204) • 

Phospho-c-Abl (Tyr245) • • 

Phospho-c-Abl (Tyr412) • • 

Phospho-c-Abl (Thr735) • 

Csk 

• 

Dexras1 

Dok1 

Cell-permeable inhibitor of PKC; activates SAPK/JNK and p38-MAPK; 

Induces apoptosis in some cell lines 

#9900 PD98059 Highly selective inhibitor of MEK1 and MEK2; binds 

inactive forms and prevent activation by upstream kinases 

#12147 PD184352 Highly selective, noncompetitive inhibitor of MEK1 and MEK2 

#8158 SB202190 Cell-permeable, highly specific inhibitor of p38-MAPK (ATP-competitive inhibitor) 

#5633 SB203580 Cell-permeable inhibitor of p38-MAPK (inhibits PDK1 at higher concentrations) 

#8705 Sorafenib Inhibits VEGFR and PDGFR; inhibits Raf kinases; induces autophagy 

#8177 SP600125 Cell-permeable, highly specific inhibitor of JNK-family kinases 

#9903 U0126 Highly selective inhibitor of MEK1 and MEK2 

• 

• 

kDa 

140 

100 

80 

60 

50 

40 

30 

20 

140 

100 

80 

60 

50 

40 

30 

20 

Phospho-JunB 

(Thr102/Thr104) 

JunB 

0 15 30 0 15 30 Anisomycin (min) 

– – – + + + SP600125 


Dok2 

• 

Phospho-Dok2 (Tyr345) • 

Phospho-DUSP1 (Ser359) • 

DUSP3/VHR 

• 

DUSP4/MKP2 

• 

DUSP6/MKP3 

• 

DUSP10/MKP5 

• 

DUSP16/MKP7 

• 

Elk-1 

• 

Phospho-Elk-1 (Ser383) • • 

Erk1 (p44 MAPK) • • 

Erk1/2 (p44/42 MAPK) • • • • 

Phospho-Erk1/2 (Thr202/Tyr204) • • • • 

Erk2 (p42 MAPK) • • 

Erk5 • • • 

Phospho-Erk5 (Thr218/Tyr220) • 

FAM129B 

• 

c-Fos • • • 

Phospho-c-Fos (Ser32) • • 

FosB • • • 

FRA1 

• 

Phospho-FRA1 (Ser265) • • • 

Phospho-FRS2-α (Tyr196) • 

Phospho-FRS2-α (Tyr436) • 


GCK 

• 

Grb2 • • 

Grb10 

• 

HGK 

• • 

JIP4/SPAG9 

• 

JNK1/SAPK 

• • • • 

Phospho-JNK1/SAPK (Thr183/Tyr185) • • • • 

JNK2 

• 

JNK3 

• 

c-Jun • • 

Phospho-c-Jun (Ser63) • • • 

Phospho-c-Jun (Ser73) • • • 

Phospho-c-Jun (Thr91) • 

Phospho-c-Jun (Thr93) • 

Phospho-c-Jun (Ser243) • 

JunB 

• • 

JunD 

• 

KSR1 

• 

Phospho-KSR1 (Ser392) • 

MAPKAPK-2 

• • 

Phospho-MAPKAPK-2 (Thr222) • 

Phospho-MAPKAPK-2 (Thr334) • • • 

MAPKAPK-3 

• • 

MAPKAPK-5 

• 

MEF2A 

• 

Phospho-MEF2A (Ser408) • 

MEF2C 

• 

MEK1 • • • • 

Phospho-MEK1 (Thr286) • 

Phospho-MEK1 (Ser298) • 

MEK1/2 • • • 

Phospho-MEK1/2 (Ser217/221) • • • • 

MEK2 • • • 

MEKK3 

• 

Mig6 

• 

MKK3 • • • 

• • 

Phospho-MKK3 (Ser189)/MKK6 

(Ser207) 

SEK1/MKK4 

• • 

Phospho-SEK1/MKK4 (Ser80) • 

Phospho-SEK1/MKK4 (Ser257) • 

Phospho-SEK1/MKK4 (Ser257/Thr261) • 

Phospho-SEK1/MKK4 (Thr261) • 

MKK6 

• • 

MKK7 • • 

Phospho-MKK7 (Ser271/Thr275) • 

MLK1 

• 

MLK3 

• 

MSK1 

• 

Phospho-MSK1 (Ser360) • 

Phospho-MSK1 (Ser376) • 

Phospho-MSK1 (Thr581) • 

MSK2 

• 

p38 MAPK • • • • 

Phospho-p38 MAPK (Thr180/Tyr182) • • • • 

p38-α MAPK • • • 

p38-β MAPK • • 


p38-δ MAPK • • • 

p38-γ MAPK • • 

PP2C α 

• 

Phospho-PTPα (Tyr798) • 

Pyk2 • • • 

Phospho-Pyk2 (Tyr402) • • 

A-Raf 

• 

Phospho-A-Raf (Ser299) • 

B-Raf • • 

Phospho-B-Raf (Ser445) • 

c-Raf • • • 

Phospho-c-Raf (Ser259) • 

Phospho-c-Raf 

(Ser289/Ser296/Ser301) 

Phospho-c-Raf (Ser296) 

Phospho-c-Raf (Ser338) 

Ras 

• 

• 

• • 

• • 

RKIP • • • 

p90RSK1 

• • • • 

Phospho-p90RSK (Thr359) • 

Phospho-p90RSK (Thr359/Ser363) • 

Phospho-p90RSK (Thr573) • 

Phospho-p90RSK (Ser380) • • • • 

RSK1/RSK2/RSK3 

• • 

RSK2 

• • 

Phospho-RSK2 (Ser227) • 

RSK3 

• 

Phospho-RSK3 (Thr356/Ser360) • 

Shc 

• 

Phospho-Shc (Tyr239/240) • 

Phospho-Shc (Tyr317) 

• 

Phospho-SHIP2 (Tyr1135) • 

SHP-2 • • • 

Phospho-SHP-2 (Tyr542) • 

Phospho-SHP-2 (Tyr580) • • • 

SOS1 

• • 

Src • • • • 

Phospho-Src (Ser17) • • 

Phospho-Src (Tyr416) 

• 

Phospho-Src (Tyr527) 

• 

Phospho-Src Family (Tyr416) • • • 

Non-phospho-Src (Tyr416) • 

Non-phospho-Src (Tyr527) • 

SRF 

• 

Phospho-SRF (Ser103) • 

TAB1 

• 

TAB2 

• • 

Phospho-TAB2 (Ser372) • 

TAK1 • • • 

Phospho-TAK1 (Thr184) • 

Phospho-TAK1 (Thr184/Thr187) • • 

Phospho-TAK1 (Thr187) 

Phospho-TAK1(Ser412) 

Tnk1 

Phospho-Tnk1 (Tyr277) 

Phospho-Tpl2/Cot (Ser400) 

• 

• 

• 

• • 

• 

745 


CST antibodies for Phospho-Erk1/2 

(Thr202/Try204) have been cited 

over 745 times in high-impact, peerreviewed 

publications from the global 

research community. 


Kusumbe, A.P. et al. (2014) Coupling 

of angiogenesis and osteogenesis 

by a specific vessel subtype in bone. 

Nature 507, 323–328. 

Brady, D.C. et al. (2014) Copper is 

required for oncogenic BRAF signalling 

and tumorigenesis. Nature 509, 

492–496. 

Dhandapany, P.S. et al. (2014) RAF1 

mutations in childhood-onset dilated 

cardiomyopathy. Nat. Genet. 46, 

635–639. 

Ota, K.T. et al. (2014) REDD1 is 

essential for stress-induced synaptic 

loss and depressive behavior. Nat. 

Med. 20, 531–535. 

von Figura, G. et al. (2014) The 

chromatin regulator Brg1 suppresses 

formation of intraductal papillary 

mucinous neoplasm and pancreatic 

ductal adenocarcinoma. Nat. Cell Biol. 

16, 255–267. 

Paul, S. et al. (2014) T cell receptor 

signals to NF-kappaB are transmitted 

by a cytosolic p62-Bcl10-Malt1-IKK 

signalosome. Sci. Signal. 7, ra45. 

Barcelo, C. et al. (2014) Phosphorylation 

at Ser-181 of oncogenic KRAS 

is required for tumor growth. Cancer 

Res. 74, 1190–1199. 

Buonato, J.M. et al. (2014) ERK1/2 

blockade prevents epithelial-mesenchymal 

transition in lung cancer cells 

and promotes their sensitivity to EGFR 

inhibition. Cancer Res. 74, 309–319. 

Cordero, J.B. et al. (2014) c-Src 

drives intestinal regeneration 

and transformation. EMBO J. 33, 

1474–1491. 

Kabekkodu, S.P. et al. (2014) DNA 

promoter methylation-dependent 

transcription of the double C2-like 

domain beta (DOC2B) gene 

regulates tumor growth in human 

cervical cancer. J. Biol. Chem. 289, 

10637–10649. 


www.cellsignal.com/cstmapk 45



MAPK/Erk in Growth and Differentiation 

Signaling Pathways Activating p38 MAP Kinase 

Ion Channels 

[Ca 2+ ] 

RTKs 

Integrins 

RTKs 

GPCR 

Inflammatory Cytokines, FasL 

DNA Damage, 

Oxidative Stress, UV 

TGF-β 

[Ca 2+ ] 

MEK1 

LAMTOR2 

MP1 

Erk1 

PLCγ 

PKC 

Ion Channels, 

Receptors 

cPLA 2 

Shc 

FRS2 

IRS 

Src 

PYK2 

GRB2 

SOS 

Spry 

Spred 

Tpl2/Cot1 

IMP 

C-TAK1 

Ras 

c-Raf 

PI3K 

PAK 

SOS 

GRB2 

Rac 

PKA 

c-Raf B-Raf 

Heterodimer 

KSR 

1/2 

MEK1/2 

Tal 

CAS RACK1 

Fyn Pax 

FAK 

[cAMP] 

B-Raf 

PD98059 

U0126 

AZD6244 

SOS 

PP1/ 

PP2At 

14-3-3 

EPAC 

Bim 

Rap1 

AMPK 

LKB1 

FMK 

SL0101 

BI-D1870 

C3G 

Raptor 

CRK 

mTOR 

Signaling 

rpS6 

eIF4B 

Filamin A 

eEF2K 

DAPK 

TCPβ 

IκBα 

GSK-3 

METTL1 

Endocytic 

Trafficking 

Gα o 

TAO1/2/3 

EEA1 

Rho-GDI 

eEF2K 

DLK 

MKK3/6 

MEKK1-4 

p38 MAPK 

TRADD 

TRAF2 

RIP 

Daxx 

MLK2/3 ASK1/2 TAK1 

MKK4 

PRAK 

Apoptosis 

SB203580 

TAB1 

HSP27 

Late Endosome 

Cell Adhesion 

Erk1/2 

Translation 

Control 

PEA-15 

p90RSK 

MNK1/2 

MKP-3 

Cytoplasmic Anchoring 

Erk1/2 

WAVE-2 

TSC2 

Migration 

p90RSK 

PLD1 

BAD 

nNos 

Ca +2 -regulated 

Exocytosis 

Translation 

MNK1/2 

cPLA 2 

Tau 

DUSPs 

p90RSK 

MAPKAPK-3 

MAPKAPK-2 

Cytokine-induced 

mRNA stability 

TNF-α biosynthesis 

Cytoplasm 

Nucleus 

MKP-1/2 

cdc25 

Erk1/2 

PPARγ 

MSK1/2 

p27 KIP1 

p90RSK 

BUB1 

Cytoplasm 

Nucleus 

p38 MAPK 

MAPKAPK-3 

PRAK 

ER 

Stat1/3 

Ets 

c-Myc/ 

N-Myc 

Elk-1 

Pax6 

TIF1A 

c-Fos 

FoxO3 

CREB 

ATF1 

Histone 

H3 

ER81 

HMGN1 

SRF 

ETV1 

c-Fos 

TIF1A 

ERα 

ATF4 

Nur77 

Myt1 

MITF 

Mad1 

C/EBPβ 

YB1 

Pax6 p53 Stat1 Max Myc Elk-1 CHOP MEF2 ATF-2 ETS1 

RARα 

MAPKAPK-2 

HMGN1 

MSK1/2 

Histone H3 

CREB 

NF-κB 

p65 

ATF1 

ER81 

UBF 

Transcription 

Transcription 

Cytokine Production, 

Apoptosis, etc. 

The MAPK/Erk signaling cascade is activated by a wide variety of receptors involved in growth and differentiation including receptor tyrosine kinases (RTKs), integrins, and ion 

channels. The specific components of the cascade vary greatly among different stimuli, but the architecture of the pathway usually includes a set of adaptors (Shc, GRB2, Crk, 

etc.) linking the receptor to a guanine nucleotide exchange factor (SOS, C3G, etc.) transducing the signal to small GTP-binding proteins (Ras, Rap1), which in turn activate the 

core unit of the cascade composed of a MAPKKK (Raf), a MAPKK (MEK1/2), and MAPK (Erk). An activated Erk dimer can regulate targets in the cytosol and also translocate to 

the nucleus where it phosphorylates a variety of transcription factors regulating gene expression. 


Anjum, R. and Blenis, J. (2008) Nat. Rev. Mol. Cell Biol. 9, 747–758. • De Luca, A., Maiello, M.R., D’Alessio, A., Pergameno, M., and Normanno, N. (2012) Expert Opin. 

Ther. 2, 17–27. • Keyse, S.M. (2008) Cancer Metastasis Rev. 27, 253–261. • Kim, E.K. and Choi, E.J. (2010) Biochim. Biophys. Acta 1802, 396–405. • Mendoza, 

M.C., Er, E.E., and Blenis, J. (2011) Trends Biochem. Sci. 36, 320–328. • Romeo, Y., Zhang, X., and Roux, P.P. (2012) Biochem. J. 441, 553–569. • Roskoski, R. Jr. 

(2012) Biochem. Biophys. Res. Commun. 417, 5–10. • Tidyman, W.E. and Rauen, K.A. (2009) Curr. Opin. Genet. Dev. 19, 230–236. 

p38 MAPKs (α, β, γ, and δ) are members of the MAPK family that are activated by a variety of environmental stresses and inflammatory cytokines. As with other MAPK 

cascades, the membrane-proximal component is a MAPKKK, typically a MEKK or a mixed lineage kinase (MLK). The MAPKKK phosphorylates and activates MKK3/6, the p38 

MAPK kinases. MKK3/6 can also be activated directly by ASK1, which is stimulated by apoptotic stimuli. p38 MAPK is involved in regulation of HSP27, MAPKAPK-2 (MK2), 

MAPKAPK-3 (MK3), and several transcription factors including ATF-2, Stat1, the Max/Myc complex, MEF-2, Elk-1, and indirectly CREB via activation of MSK1. 


Coulthard, L.R., White, D.E., Jones, D.L., McDermott, M.F., and Burchill, S.A. (2009) Trends Mol. Med. 15, 369–379. • Cuadrado, A. and Nebreda, A.R. (2010) Biochem. J. 

429, 403–417. • del Barco Barrantes, I. and Nebreda, A.R. (2012) Biochem. Soc. Trans. 40, 79–84. • Huang, G., Shi, L.Z., and Chi, H. (2009) Cytokine 48, 161–169. • 

Kostenko, S., Dumitriu, G., Lægreid, K.J., and Moens, U. (2011) World J. Biol. Chem. 2, 73–89. • Shiryaev, A. and Moens, U. (2010) Cell. Signal. 22, 1185–1192. 

© 2004–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. John Blenis, Harvard Medical School, Boston, MA, for reviewing this diagram. 





SAPK/JNK Signaling 

Growth Factors, UV 

RTKs 

CrkII 

CrkL 

Shc 

GRB2 

HPK1 

Cytoplasm 

Nucleus 

PI3K 

14-3-3 

SOS 

MAPKKKs : 

TAK1 

Ras 

MKK4/7 

JNK1/2/3 

IRS-1 

c-Abl 14-3-3 14-3-3 

DUSPs 

c-Abl 

Cellular Stress, 

γ Radiation 

Rac 

MEKK1/4 

MLK2/3 

DLK 

TpI-2 

TAO1/2 

NO 

Cdc42 

Rho 

MLKs 

POSH 

JNK 

JNK 

FasL, UV 

Inflammatory Cytokines 

GCK 

ASK1 

Rac 

c-Jun ATF-2 Elk-1 Smad4 p53 NFAT4 NFATc1 Stat3 

TRADD 

TRAF2 

JIP1,2,3 

RIP 

Daxx 

Transcription 

Oxidative 

Stress 

Apoptosis 

Cdc42 

MAPKKKs 

Bax 

HSF1 

Gα 12/13 

14-3-3 

Bax 

Growth 


Survival 

Apoptosis 

G 12/13 -coupled 

Receptors 

Gβγ 

PI3Kγ 

Mitochondrial 

Translocation/ 

Apoptosis 

Stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) are members of the MAPK family and are activated by a variety of environmental stresses, inflammatory 

cytokines, growth factors, and GPCR agonists. Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42). As with the other 

MAPKs, the membrane proximal kinase is a MAPKKK, typically MEKK1–4, or a member of the mixed lineage kinases (MLK) that phosphorylates and activates MKK4 (SEK) or 

MKK7, the SAPK/JNK kinases. Alternatively, MKK4/7 can be activated by a member of the germinal center kinase (GCK) family in a GTPase-independent manner. SAPK/JNK 

translocates to the nucleus where it can regulate the activity of multiple transcription factors. 


Bogoyevitch, M.A., Ngoei, K.R., Zhao, T.T., Yeap, Y.Y., and Ng, D.C. (2010) Biochim. Biophys. Acta. 1804, 463–475. • Chen, F. (2012) Cancer Res. 72, 379–386. • Davies, 

C. and Tournier, C. (2012) Biochem. Soc. Trans. 40, 85–89. • Engström, W., Ward, A., and Moorwood, K. (2010) Cell Prolif. 43, 56–66. • Haeusgen, W., Herdegen, T., and 

Waetzig, V. (2011) Eur. J. Cell Biol. 90, 536–544. • Verma, G., and Datta, M. (2012) J. Cell Physiol. 227, 1791–1795. 

PI3 Kinase/Akt Signaling 

Activation of Akt 

The serine/threonine kinase Akt/PKB exists as three isoforms in mammals. Akt1 and Akt2 have a wide 

tissue distribution, whereas Akt3 is expressed in testes and brain. Akt regulates multiple biological processes 

including cell survival, proliferation, growth, and glycogen metabolism. Various growth factors, 

hormones, and cytokines activate Akt by binding their cognate receptor tyrosine kinase (RTK), cytokine 

receptor, or G protein-coupled receptor (GPCR) and triggering activation of the lipid kinase PI3K, which 

generates PIP 3 at the plasma membrane. Akt binds PIP 3 through its pleckstrin homology (PH) domain, 

resulting in translocation of Akt to the membrane. Akt is activated through a dual phosphorylation 

mechanism. PDK1, which is also brought to the membrane through its PH domain, phosphorylates Akt 

within its activation loop at Thr308. A second phosphorylation at Ser473 within the C-terminus is also 

required for activity and is carried out by the mTOR-Rictor complex, mTORC2. 

Insulin stimulation results in clustering of phospho-Akt at the membrane. 

Phospho-Akt (Thr308) (D25E6) XP ® Rabbit 

mAb #13038: Confocal IF analysis of C2C12 

cells, insulin-treated (100 nM, 15 min; left) or 

treated with LY294002 #9901 (50 μM, 2 hr; 

right), using #13038 (green). Actin filaments 

were labeled with DY-554 phalloidin (red). Blue 


DNA dye). 

Growth factor stimulation results in phosphorylation 

of Akt at Thr308, one of two phosphorylations 

necessary for Akt activation. 

Phospho-Akt (Thr308) (D25E6) XP ® Rabbit mAb #13038: Western blot analysis of 

extracts from NIH/3T3 cells, untreated or treated with Human Platelet-Derived Growth 

Factor AA (hPDGF-AA) #8913 (100 ng/ml, 5 min; +), and untreated LNCaP and PC-3 

cells, using #1308 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower). 

Lanes 

1. NIH/3T3 

2. NIH/3T3 

3. LNCaP 

4. PC-3 

kDa 

140 

100 

80 

60 

50 

60 

50 

1 2 3 4 

Phospho- 

Akt (Thr308) 

Akt (pan) 

– + – – hPDGF 

Inhibition of Akt Activity 

PTEN, a lipid phosphatase that catalyzes the dephosphorylation of PIP 3 , is a major negative regulator of 

Akt signaling. Loss of PTEN function has been implicated in many human cancers. Akt activity is also 

negatively regulated by the phosphatases PP2A and PHLPP, as well as by the chemical modulators 

wortmannin and LY294002, both of which are inhibitors of PI3K. 

PTEN expression in human colon 

PTEN (D4.3) XP ® Rabbit mAb #9188: IHC analysis 

of paraffin-embedded human colon using #9188. 

kDa 

100 


Akt-activating treatments 

and absence 

of PTEN result in 

phosphorylation of 

Akt at Ser473. 

A 

80 

60 

50 

80 

60 

50 

PC-3 

– + 

– + 

– – 

NIH/3T3 

– – 

– – 

– + 

Phospho-Akt (Ser473) 

is expressed in human 

lung carcinoma. 

B 

Phospho- 

Akt (Ser473) 

Akt 

wortmannin 

LY294002 

PDGF 

Phospho-Akt (Ser473) (D9E) XP ® 

Rabbit mAb #4060: WB analysis of 

extracts from PC-3 cells (PTEN null), 

untreated or treated with LY294002 

#9901/Wortmannin #9951, and NIH/3T3 

cells, serum-starved or PDGF-treated 

(A), using #4060 (upper) or Akt (pan) 

(C67E7) Rabbit mAb #4691 (lower). IHC 


lung carcinoma (B) using #4060. 



www.cellsignal.com/cstakt 49



Akt Downstream Signaling 

Activated Akt phosphorylates a large number of downstream substrates containing the consensus sequence 

RXRXXS/T. One of its primary functions is to promote cell growth and protein synthesis through 

regulation of the mTOR signaling pathway. Akt directly phosphorylates and activates mTOR, as well as 

inhibits the mTOR inhibitor proteins PRAS40 and tuberin (TSC2). Combined, these actions promote cell 

growth and G1 cell cycle progression through signaling via p70 S6 Kinase and inhibition of 4E-BP1. 

GSK-3 is a primary target of Akt and inhibitory phosphorylation of GSK-3α (Ser21) or GSK-3β (Ser9) 

has numerous cellular effects such as promoting glycogen metabolism, cell cycle progression, regulation 

of Wnt signaling, and contributing to the formation of neurofibrillary tangles in Alzheimers disease. 

Akt promotes cell survival directly by its ability to phosphorylate and inactivate several pro-apoptotic 

targets, including Bad, Bim, Bax, and the Forkhead (FoxO1/3a) transcription factors. Akt also plays an 

important role in metabolism and insulin signaling. Insulin receptor signaling through Akt promotes 

Glut4 translocation through activation of AS160 and TBC1D1, resulting in increased glucose uptake. 

Akt regulates glycolysis through phosphorylation of PFK and hexokinase, and plays a significant role in 

aerobic glycolysis in cancer cells, also known as the Warburg Effect. 

Akt in Disease 

Aberrant Akt signaling is the underlying defect found in several pathologies. Akt is one of the most 

frequently activated kinases in human cancer as constitutively active Akt can promote unregulated 

cell proliferation. Abnormalities in Akt2 signaling can result in diabetes due to defects in glucose 

homeostasis. Akt is also a key player in cardiovascular disease through its role in cardiac growth, 

angiogenesis, and hypertrophy. 


Bar-Peled, L. and Sabatini, D.M. (2014) Trends Cell Biol. 24, 400−406. • Correia, N.C., Gírio, A., Antunes, I., et al. (2014) 

Eur. J. Cancer 50, 216−225. • Betz, C. and Hall, M.N. (2013) J. Cell. Biol. 203, 563−574. • Fruman, D.A. and Rommel, C. 

(2014) Nat. Rev. Drug Discov. 13, 140−156. • Kumar, A., Rajendran, V., Sethumadhavan, R., and Purohit, R. (2013) Scientific 

World Journal 2013, 756134. • Ortega-Molina, A. and Serrano, M. (2013) Trends Endocrinol. Metab. 24, 184−189. • Porta, 

C., Paglino, C., and Mosca, A. (2014) Front Oncol. 4, 64. • Shimobayashi, M. and Hall, M.N. (2014) Nat. Rev. Mol. Cell Biol. 

15, 155−162. • Toker, A. and Marmiroli, S. (2014) Adv. Biol. Regul. 55, 28−38. 

Inhibitors of 

PI3 Kinase/Akt/mTOR 

#12017 Everolimus 

Specific inhibitor of 

mTORC1 protein complex 

#9904 Rapamycin 

Specific inhibitor of 

mTORC1 protein complex 

#9901 LY294002 

Reversible but potent 

inhibitor of PI3K family 

members 

#9951 Wortmannin 

Irreversible, potent inhibitor 

of PI3K family members 

Akt activation by insulin 

treatment results in 


mTOR at Ser2448. 

kDa 

200 

140 

100 

80 

200 

140 

100 

80 

1 2 3 

Phospho-mTOR 

(Ser2448) 

mTOR 

Phospho-mTOR (Ser2448) (D9C2) 

XP ® Rabbit mAb #5536: WB analysis 

of extracts from serum-starved NIH/3T3 

cells, untreated or insulin-treated (150 

nM, 5 min), alone or in combination with 

λ-phosphatase, using #5536 (upper) or 

mTOR (7C10) Rabbit mAb #2983. 

Lanes 

1. untreated 

2. insulin-treated (150 nM, 5 min) 

3. λ-phosphatase and insulin-treated 

(150 nM, 5 min) 

Inhibitors of Akt activation prevent phosphorylation of GSK-3β at Ser9. 

Phospho-GSK-3β (Ser9) (D85E12) XP ® Rabbit mAb #5558: Confocal IF analysis of PC-3 cells, untreated (A), LY294002- and 

Wortmannin-treated (#9901 and #9951 respectively) (B), or λ phosphatase-treated (C), using #5558 (green). Actin filaments were labeled 

with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). 

Active Akt phosphorylates FoxO3a at Ser253, retaining 

FoxO3a in the cytoplasm and preventing cell death. 

A 

kDa 

140 

100 

80 

60 

50 

140 

100 

80 

1 2 

Phospho-FoxO3 

(Ser253) 

FoxO3 

Lanes 

1. untreated 

2. treated overnight with 

LY294002 #9901 (50 μM) and 

Wortmannin #9951 (1 μM) 

A 

B 

B 

Phospho-FoxO3a (Ser253) (D18H8) Rabbit mAb #13129: WB analysis of extracts from 

293T cells (A) using #13129 (upper) or FoxO3a (D19A7) Rabbit mAb #12829 (lower). 

FoxO3a (75D8) Rabbit mAb #2497: Confocal IF analysis of SH-SY5Y cells, IGF-I treated 

(B) or LY294002-treated (C), using FoxO3a (75D8) Rabbit mAb (green). Actin filaments were 

labeled with Alexa Fluor ® 555 Phalloidin #8953 (red). Blue pseudocolor = DRAQ5 ® #4084 


Akt Signaling Research 

Please visit our website for additional resources and products relating to the study of Akt Signaling. 

www.cellsignal.com/cstakt 

C 

C 

Commonly Studied Akt Targets 


Akt1 • • • 

Phospho-Akt1 (Ser129) • 

Phospho-Akt1 (Ser473) • • 

Phospho-Drosophila Akt (Ser505) • 

Akt1/Akt2/Akt3 • • • • 

Phospho-Akt1 (Thr308)/Phospho-Akt2 

(Thr309)/Phospho-Akt3 (Thr305) • • • • 

Phospho-Akt1 (Ser473)/Phospho-Akt2 

(Ser474)/Phospho-Akt3 (Ser472) • • • • 

Phospho-Akt1 (Thr450)/Phospho-Akt2 

(Thr451)/Phospho-/Akt3 (Thr447) • • 

Akt2 • • • • 

Phospho-Akt2 (Ser474) • • 

Akt3 

• • • 

Phospho-Akt3 (Ser472) 

• 

ATP6V1B1/2 

• 

CTMP 

• 

DEPTOR/DEPDC6 

• 

DJ-1 

• • 

eNOS 

• • 

Phospho-eNOS (Ser113) • 

Phospho-eNOS (Thr495) • 

Phospho-eNOS (Ser1177) • • • 

FKBP5 

• • 

FLCN 

• 

FoxO1 • • • • 

• 

Phospho-FoxO1 (Thr24)/FoxO3a 

(Thr32) 

Phospho-FoxO1 (Thr24)/FoxO3a 

(Thr32)/FoxO4 (Thr28) 

Phospho-FoxO1 (Ser256) 

Phospho-FoxO1 (Ser319) 

• 

• 

• 

FoxO3a • • 

Phospho-FoxO3a (Ser253) • • 

Phospho-FoxO3a (Ser294) • 

Phospho-FoxO3a (Ser318/Ser321) • 

Phospho-FoxO3a (Ser413) • 

FoxO4 

• • 

Phospho-FoxO4 (Ser193) • 

Gab1 

• 


Phospho-Gab1 (Tyr307) • 

Phospho-Gab1 (Tyr627) • • 

Gab2 

• 

Phospho-Gab2 (Ser159) • 

Phospho-Gab2 (Tyr452) 

• • 

GβL • • • 

GSK-3α • • • 

Phospho-GSK-3α (Ser21) • 

GSK-3α/GSK-3β • • 

Phospho-GSK-3α/β (Ser21/9) • 

GSK-3β • • 

Phospho-GSK-3β (Ser9) • • • 

Phospho-GSK-3β (Thr390) 

HGS 

LAMTOR1/C11orf59 

LAMTOR2/ROBLD3 

LAMTOR3/MAPKSP1 

LAMTOR4/C7orf59 

MAP4K3 

Mios 

mTOR 

Phospho-mTOR (Ser2448) 

Phospho-mTOR (Ser2481) 

p70 S6 Kinase 

Phospho-p70 S6 Kinase (Ser371) 

Phospho-p70 S6 Kinase (Thr389) 

Phospho-p70 S6 Kinase 

(Thr421/Ser424) 

Phospho-Drosophila p70 S6 

Kinase (Thr398) 

• 

• 

• • 

• 

• 

• 

• 

• 

• • • • • 

• • 

• • 

• • • • • 

• 

• • • 

• 

• 

PDK1 • • • 

Phospho-PDK1 (Ser241) • • 

PI3 Kinase p55 

• 

PI3 Kinase p85 α • • 


• • 

Phospho-PI3 Kinase p85 

(Tyr458)/p55 (Tyr199) 

• 


• 

PI3 Kinase p110 α • • • 


nodes within Akt Signaling pathways 

and are commonly studied in Akt 

research. Primary antibodies, antibody 

conjugates, and antibody sampler kits 

containing these targets are available 

from CST. 










51

Other 




PI3 Kinase p110 β • • 

PI3 Kinase p110 γ • • 

PI4 Kinase 

• 

PI3 Kinase Class II α • 

PI3 Kinase Class III • • 

PNUTS 

• 

PRAS40 • • • • 

Phospho-PRAS40 (Ser183) • 

Phospho-PRAS40 (Thr246) • • • 

Protor2 

• 

PTEN 

• • • • • 

Phospho-PTEN (Ser380) • • 

Phospho-PTEN (Ser380/Thr382/383) • • 

non-Phospho-PTEN 

(Ser380/Thr382/Thr383) • • 

RagA 

• 

RagB 

• 

RagC 

• • 

RagD 

• 

Raptor • • 

Phospho-Raptor (Ser792) • 

REDD1 

• 

Rheb 

• 

Rictor • • • • 


Phospho-Rictor (Thr1135) • 

SGK1 

• 

Phospho-SGK1 (Ser78) • • 

SGK2 

• • 

SGK3 

• 

Phospho-SGK3 (Thr320) • 

Sin1 

• 

TCL1 

• • 

Hamartin/TSC1 

• • 

Tuberin/TSC2 • • • 

Phospho-Tuberin/TSC2 (Ser939) • 



Phospho-Tuberin/TSC2 (Thr1462) • • 

Phospho-Tuberin/TSC2 (Tyr1571) • 

WNK1 

• 

Phospho-WNK1 (Thr60) • 

WNK4 

• 

YAP 

• • 

Phospho-YAP (Ser127) • • 

Phospho-YAP (Ser397) • 

YB1 • • • 

Phospho-YB1 (Ser102) • • 


Protein Synthesis 

FAK 

Paxillin 

S6 

FoxO1 

PI3K 

p70 S6K 

PDCD4 

Inhibits 

Autophagy 

Bim 

ILK 

Bcl-2 

Integrin 

4E- 

BP1 

ATG13 

Inhibits Apoptosis 

TRAF6 

mTORC1 

XIAP 

PI3K 

Gab1 

Gab2 

ATM/ATR 

DNA-PK 

PTEN Akt 

PIP 

PIP 3 

3 

p53 

IRS-1 

TSC2 

TSC1 

PRAS40 

RTK 

NEDD1-4 

TBK1 

Membrane PDK1 

Recruitment 

and Activation 

MDM2 

Bad 

14-3-3 

DNA Damage 

Jak1 

Akt 

Cytokine 

Receptor 

PI3K 

mTORC2 

PDK1 

GSK-3 

CD19 

PI3K 

Membrane 

Recruitment 

and Activation 

mIg 

BCR 

BCAP 

Syk 

PFKFB2 

Ag 

α/β α/β 

PIP5K AS160 

Lyn 

p47phox 

Lamin A 

Tpl2 

IKKα 

eNOS 

PI3K 

Gβγ 

Hexokinase II 

Gα 

GTP 

PTEN PP2A PHLPP1/2 CTMP 

Huntingtin 

Ataxin-1 

mIg 

GABAAR 

GPCR 

Neutrophil 

Respiratory 

Burst 

Organization 

of Nuclear 

Proteins 

NF-κB 

Pathway 

Cardiovascular 

Homeostasis 

Synaptic 

Signaling 

Blocks 

Aggregation 

Promotes 

Neuroprotection 

Aggregation 

Neurodegeneration 

Glycolysis 

Neuroscience 

996 


CST antibodies for Phospho-Akt 

(Ser473) have been cited over 996 times 

in high-impact, peer-reviewed publications 



Liu, P. et al. (2014) Cell-cycle-regulated activation of Akt 

kinase by phosphorylation at its carboxyl terminus. Nature 

508, 541–545. 

Ardestani, A. et al. (2014) MST1 is a key regulator of beta 

cell apoptosis and dysfunction in diabetes. Nat. Med. 20, 

385–397. 

White, A.C. et al. (2014) Stem cell quiescence acts as a 

tumour suppressor in squamous tumours. Nat. Cell Biol. 

16, 99–107. 

Snuderl, M. et al. (2013) Targeting placental growth factor/ 

neuropilin 1 pathway inhibits growth and spread of medulloblastoma. 

Cell 152, 1065–1076. 

Hirabayashi, S. et al. (2013) Transformed Drosophila cells 

evade diet-mediated insulin resistance through wingless 

signaling. Cell 154, 664–675. 

Yan, L. et al. (2013) The ubiquitin-CXCR4 axis plays an 

important role in acute lung infection-enhanced lung tumor 

metastasis. Clin. Cancer Res. 19, 4706–4716. 

Keysar, S.B. et al. (2013) Hedgehog signaling alters reliance 

on EGF receptor signaling and mediates anti-EGFR 

therapeutic resistance in head and neck cancer. Cancer Res. 

73, 3381–3392. 

Romaker, D. et al. (2014) MicroRNAs are critical regulators 

of tuberous sclerosis complex and mTORC1 activity in the 

size control of the Xenopus kidney. Proc. Natl. Acad. Sci. 

USA 111, 6335–6340. 

Nguyen, A. et al. (2014) Very low density lipoprotein receptor 

(VLDLR) expression is a determinant factor in adipose tissue 

inflammation and adipocyte-macrophage interaction. J Biol. 

Chem. 289, 1688–1703. 

Xiang, M. (2014) STAT3 induction of miR-146b forms a 

feedback loop to inhibit the NF-kappaB to IL-6 signaling axis 

and STAT3-driven cancer phenotypes. Sci Signal. 7, ra11. 

Sakr, R.A. et al. (2014) PI3K pathway activation in highgrade 

ductal carcinoma in situ–implications for progression 

to invasive breast carcinoma. Clin. Cancer Res. 20, 

2326–2337. 

Wong, C.C. et al. (2014) Inactivating CUX1 mutations 

promote tumorigenesis. Nat. Genet 46, 33–38. 

Lee, E.Y. and Abbondante, S. (2014) Tissue-specific tumor 

suppression by BRCA1. Proc. Natl. Acad. Sci. USA 111, 

4353–4354. 

Li, H. et al. (2014) Lysophosphatidic acid acts as a nutrientderived 

developmental cue to regulate early hematopoiesis. 

EMBO J. 33, 1383–1396. 

Stanton, M.J. et al. (2013) Autophagy control by the VEGF- 

C/NRP-2 axis in cancer and its implication for treatment 

resistance. Cancer Res. 73, 160–171. 

Wu, W. et al. (2013) Inhibition of tumor growth and metastasis 

in non-small cell lung cancer by LY2801653, an inhibitor 

of several oncokinases, including MET. Clin. Cancer Res. 19, 

5699–5710. 

Mercan, F. et al. (2013) Novel role for SHP-2 in nutrientresponsive 

control of S6 kinase 1 signaling. Mol. Cell Biol. 

33, 293–306. 

Survival 

mTORC1 

GβL Raptor 

mTOR 

DEPTOR 

Bax 

Apoptosis 

Cytosolic 

Sequestration 

Palladin 

Girdin 

Migration 

Migration 

Wee1 

CDK2 Cyclin A 

Skp2 SCF 

Myt1 

p27 Kip1 

Cell Cycle 

Proliferation 

p21 

Waf1/Cip1 

GS 

Cyclin D1 

ATP-Citrate 

Lyase 

Fatty Acid 

Synthesis 

Glycogen 

Synthesis 

Glucose 

Transport 

Glucose Metabolism 

mTORC2 

Sin1 PRR5 

Rictor GβL 

mTOR 

DEPTOR 

Since its initial discovery as a proto-oncogene, the serine/threonine kinase Akt (also known as protein kinase B or PKB) has become a major focus of attention because of 

its critical role in regulating diverse cellular functions including metabolism, growth, proliferation, survival, transcription and protein synthesis. The Akt signaling cascade is 

activated by receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G-protein-coupled receptors and other stimuli that induce production of phosphatidylinositol 

(3,4,5) trisphosphates (PIP3) by phosphoinositide 3-kinase (PI3K). These lipids serve as plasma membrane docking sites for proteins that harbor pleckstrin-homology 

(PH) domains, including Akt and its upstream activator PDK1. At the membrane PDK1 phosphorylates Akt at Thr308 leading to partial activation of Akt. Phosphorylation of 

Akt at Ser473 by mTORC2 stimulates full enzymatic activity. Members of the PI3K-related kinase (PIKK) family, including DNA-PK, can also phosphorylate Akt at Ser473. Akt 

is dephosphorylated by protein phosphatase 2A (PP2A) and the PH-domain leucine-rich-repeat-containing protein phosphatases (PHLPP1/2). In addition, the tumor suppressor 

phosphatase and tensin homolog (PTEN) inhibits Akt activity by dephosphorylating PIP3. 

Dysregulation of the PI3K/Akt pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. In cancer, 

two mutations that increase the intrinsic kinase activity of PI3K have been identified. In addition, PTEN is frequently mutated or lost in human tumors. Activating mutations 

in Akt have also been described. The frequency with which dysregulated Akt signaling contributes to human disease has culminated in the aggressive development of small 

molecule inhibitors of PI3K and Akt. 

There are three highly related isoforms of Akt (Akt1, Akt2 and Akt3), which phosphorylate substrates containing the consensus phosphorylation motif RxRxxS/T. Akt isoforms 

share many substrates but isoform-specific Akt substrates have also been identified. For example, all Akt isoforms are able to phosphorylate PRAS40 (proline-rich Akt substrate 

of 40 kDa) but only Akt1 can phosphorylate the actin-associated protein palladin. 

Akt regulates cell growth through its effects on the TSC1/TSC2 complex and mTORC signaling. Akt contributes to cell proliferation via phosphorylation of the CDK inhibitors 

p21 and p27. Akt is a major mediator of cell survival through direct inhibition of pro-apoptotic proteins like Bad or inhibition of pro-apoptotic signals generated by transcription 

factors like FoxO. Akt is critically involved in the regulation of metabolism through activation of AS160 and PFKFB2. In addition, Akt has been shown to regulate proteins 

involved in neuronal function including the GABA receptor, ataxin-1 and huntingtin proteins. Akt contributes to cell migration and invasion via phosphorylation of palladin and 

vimentin. Akt also regulates NF-κB signaling by phosphorylating IKKα and Tpl2. Due to the critical role of Akt/PKB in regulating diverse cellular functions it is an important 

therapeutic target for the treatment of human disease. 


Bhaskar, P.T. and Hay, N. (2007) Dev. Cell 12, 487–502. • Bozulic, L. and Hemmings, B.A. (2009) Curr. Opin. Cell Biol. 21, 256–261. • Brugge, J., Hung, M.C., and Mills, 

G.B. (2007) Cancer Cell 12, 104–107. • Carnero, A., Blanco-Aparicio, C., Renner, O., Link, W., and Leal, J.F. (2008) Curr. Cancer Drug Targets 8, 187–198. • Hers, I., 

Vincent, E.E., and Tavaré, J.M. (2011) Cell Signal. 23, 1515–1527. • Liu, P., Cheng, H., Roberts, T.M., and Zhao, J.J. (2009) Nat. Rev. Drug Discov. 8, 627–644. • Manning, 

B.D. and Cantley, L.C. (2007) Cell 129, 1261–1274. • Salmena, L., Carracedo, A., and Pandolfi, P.P. (2008) Cell 133, 403–414. 

© 2007–2015 Cell Signaling Technology, Inc. • We would like to thank Kristin Brown and Prof. Alex Toker, Beth Israel Deaconess Medical Center, Harvard Medical School for reviewing this diagram. 





mTOR Signaling 

GRB10 

Sin1 

Torin1 

PP242 

KU63794 

WYE354 

PRR5 

mTORC2 

Rictor GβL 

mTOR 

DEPTOR 

SGK1 

rapamycin 

FKBP12 

PKCα 

PRAS40 

p70S6K 

Cell Growth 

IRS-1 

Akt 

TSC1 

TSC2 

TBC1D7 

Rheb 

mTORC1 

GβL 

Raptor 

mTOR 

DEPTOR 

FIP200 

Atg13 

ULK 

Glucose 

PIP 3 PIP 2 

AMP: ATP 


Hormones, 

AICAR 

Cytokines, etc. 

Stress 

Hypoxia 

PI3K 

Dvl 

DNA 

Damage 

PDK1 

Ras 

PTEN 

p53 

Autophagy 

eIF4G 

GATOR2 


4E- 

BP1/2 

mRNA 

Translation 

Proliferation 

LRP 

Erk 

RSK 

GSK-3 

REDD1/2 

AMPK 

Lipin 1 

Wnt 

Frizzled 

Gα q/o 

mTORC1 

Translocation 

to Lysosome 

Lipid Synthesis 

LKB1 

Sestrin-1/2 

LAMTOR 

1/2/3/4/5 

Ragulator 

Complex 

Rag A/B 

GTP 

Rag C/D 

GDP 

Ribosome 

Biogenesis 

TFEB 

PPARα 

HIF-1 

PGC-1α 

PPARγ 

SKAR 

mRNA Splicing 

metformin 

Glucose, 

Amino Acids 

V-ATPase 

Mios 

WDR59 

Lipogenesis 

SREBP-1 

Transcription 

Seh1L 

WDR24 

Sec13 

GATOR1 

DEPDC5 Nprl2 

Nprl3 

Lipid 

Metabolism 

FLCN 

FNIP1/2 

Autophagy/Lysosome 

Biogenesis 

VEGF/ 

Angiogenesis 

Mitochondrial 

Metabolism 

Adipogenesis 

The mechanistic target of rapamycin (mTOR) is an atypical serine/threonine kinase that is present in two distinct complexes. The first, mTOR complex 1 (mTORC1), is 

composed of mTOR, Raptor, GβL, and DEPTOR and is inhibited by rapamycin. It is a master growth regulator that senses and integrates diverse nutritional and environmental 

cues, including growth factors, energy levels, cellular stress, and amino acids. It couples these signals to the promotion of cellular growth by phosphorylating substrates 

that potentiate anabolic processes such as mRNA translation and lipid synthesis, or limit catabolic processes such as autophagy. The small GTPase Rheb, in its GTP-bound 

state, is a necessary and potent stimulator of mTORC1 kinase activity, which is negatively regulated by its GAP, the tuberous sclerosis heterodimer TSC1/2. Most upstream 

inputs are funneled through Akt and TSC1/2 to regulate the nucleotide-loading state of Rheb. In contrast, amino acids signal to mTORC1 independently of the PI3K/Akt axis to 

promote the translocation of mTORC1 to the lysosomal surface where it can become activated upon contact with Rheb. This process is mediated by the coordinated actions of 

multiple complexes, notably the v-ATPase, Ragulator, the Rag GTPases, and GATOR1/2. The second complex, mTOR complex 2 (mTORC2), is composed of mTOR, Rictor, GβL, 

Sin1, PRR5/Protor-1, and DEPTOR. mTORC2 promotes cellular survival by activating Akt, regulates cytoskeletal dynamics by activating PKCα, and controls ion transport and 

growth via SGK1 phosphorylation. Aberrant mTOR signaling is involved in many disease states including cancer, cardiovascular disease, and diabetes. 


Dowling, R.J., Topisirovic, I., Fonseca, B.D., and Sonenberg, N. (2010) Biochim. Biophys. Acta. 1804, 433–439. • Dunlop, E.A. and Tee, A.R. (2009) Cell. Signal. 21, 

827–835. • Hoeffer, C.A. and Klann, E. (2010) Trends Neurosci. 33, 67–75. • Laplante, M. and Sabatini, D.M. (2013) J. Cell Sci. 126, 1713–1719. • Laplante, M. and 

Sabatini, D.M. (2012) Cell 149, 274–293. • Neufeld, T.P. (2010) Curr. Opin. Cell Biol. 22, 157–168. • Zoncu, R., Efeyan, A., and Sabatini, D.M. (2011) Nat. Rev. Mol. Cell 

Biol. 12, 21–35. 


Akt Substrates 

MAPK, mTOR, and the PI3K/Akt pathways are key signaling pathways activated downstream of oncogenic receptor tyrosine 

kinases (RTKs). All of these pathways activate AGC kinase family members, including Akt, RSK, and p70 S6 kinases, whose 

protein substrates are phosphorylated at the RxRxxS/T motif. 

In a phosphoproteomic study co-authored by scientists in the Cell Signaling Technology (CST) Site Discovery Group (Moritz, A. 

et al. (2010) Sci. Signal 24,ra64), over 300 novel downstream substrates for these AGC family kinases were identified. The 

experimental approach involved the use of PhosphoScan ® , CST’s proprietary methodology for antibody-based peptide enrichment 

combined with tandem mass spectrometry for quantitative profiling of post-translational modifications. A key step was 

the development of a RxRxxS/T motif antibody, which was then used as an affinity reagent to selectively immunoprecipitate 

phosphorylated substrates of Akt, RSK, and p70 S6 kinases. The antibody was employed in PhosphoScan in three different 

cancer cell lines, dependent on either EGFR, c-Met, or PDGFR, allowing mapping of the signaling network downstream of these 

RTKs. Substrates included proteins involved in many cellular functions, including scaffolding, protein stability, metabolism, trafficking, 

and motility. 

Substrate 

GAP/GEF/Adaptors 

ARHGAP19 

ARHGEF12 

AS250 

TBC1D1 

TBC1D4 

Receptors/Transporters 

DR6 SLC20A2 

EPHA2 SLC9A1 

FGFR2 

SEMA4B 

TSC2 

CD2AP 

FRS2 

IRS1 

IRS2 

Adhesion/Cytoskeleton 

DSP PLEC1 

MLLT4 SVIL 

KIF21A PPP1R12A 

AMPKA 

Isoform 

RICTOR 

CABLES1 

LMO7 

Kinases 

WNK1 

PKD2 

HGK 

BRD2 

UO126 

EGFR 

Gefitinib Su11274 Gleevec 

Ras 

MEK 

RSK 

Energy/ 

Metabolism 

PANK2 

PFKFB2 

OXR1 

GSK-3α 

GSK-3β 

PI3K 

Akt 

Met 

SGKs 

mTOR 

Wort 

S6K 

PDGFRα 

RNA Processing/ 

Translation 

LARP1 

MEPCE 

RPS6 

EIF4ENIF1 

EDC3 

Published Data Human 

Organism Site Site Sequence (+/-7) PMID 

Rapa 

Chaperone/Ubiquitin 

CCT2 

DNAJC2 

SGTA 

NEDD4-2 

UBR4 

UBXN4 

NIPA 

TIF1-γ 

Survival 

AKT1S1 

BAD 

NDRG2 

NDRG3 

Transcription 

FOXO3 

GTF3C1 

IWS1 

TAF3 

TCF12 

Vesicle Trafficking 

C4orf16 REPS1 

GOLGA4 STX12 

NDRG1 STX7 

HDGF2 

TCF3 

BRD1 

SP100 

Substrate Function and 

Effect of Phosphorylation 

14-3-3 z Akt1 human S58 S58 VVGARRSsWRVVssI 11956222 A key regulatory protein in signal transduction, 

checkpoint control, apoptotic, 

and nutrient-sensing pathways; effect 

of phosphorylation is unknown 

acinus Akt1 human S1180 S1180 GPRsRsRsRDRRRKE 18559500, 

16177823 

Akt1 rat S1329 S1331 HSRSRSRsTPVRDRG 16177823 

Induces chromatin condensation during 

apoptosis; phosphorylation inhibits 

this process 

ACLY Akt1 mouse S455 S455 PAPSRtAsFsESRAD 16007201 Catalyzes the formation of acetyl-CoA 

and oxaloacetate (OAA) in the cytosol; 

phosphorylation enhances the catalytic 

activity of the enzyme 

ADRB2 Akt1 human S346 S346 LLCLRRssLKAyGNG 11809767 A receptor that binds epinephrine 

and norepinephrine, acting as a 

neuromodulator in the central nervous 

system and as a hormone in the 

vascular system; phosphorylation in 

response to insulin stimulation leads to 

sequestration of ADBR2 

Akt1 Akt1 human S246, 

T72 

S246, 

T72 

LSRERVFsEDRARFY, 

TERPRPNtFIIRCLQ 

Akt1 mouse S473 S473 RPHFPQFsYsAsGtA 11570877, 

10722653 

16549426 Activated by insulin and various growth 

and survival factors to function in a 

wortmannin-sensitive PI3 kinaseinvolved 

pathway controlling survival 

and apoptosis; autophosphorylation 

activates the kinase 

AMPKA1 Akt1 rat S485 S485 ATPQRSGsISNYRSC 16340011 Heterotrimeric complex that plays a 

key role in the regulation of energy 

homeostasis; phosphorylation regulates 

AMPK activity 

AMPKA2 Akt1 rat S491 S491 STPQRSCsAAGLHRP 16340011 Heterotrimeric complex that plays a 

key role in the regulation of energy 

homeostasis; phosphorylation regulates 

AMPK activity 




Substrate 

AMPKA 

Isoform 





APS Akt1 rat S588 S598 SARSRSNsTEHLLEA 16141217 An adaptor protein recruited to the insulin 

receptor to signal insulin-stimulated 

glucose transport; phosphorylation 

promotes membrane localization 

AR Akt1 human S213, 

S791 

S213, 

S791 

SGRAREAsGAPTsSK, 

CVRMRHLsQEFGWLQ 

11404460, 

14555644, 

17470458, 

11156376 

Nuclear receptor; phosphorylation 

suppresses AR activation, expression 

of AR target genes, and AR-mediated 

apoptosis 

arfaptin 2 Akt1 human S260 S260 GTRGRLEsAQATFQA 15809304 ADP ribosylation factor-interacting 

protein, implicated as a factor in 

Huntington's disease; phosphorylation 

promotes neuronal cell survival and 

neuroprotection 

ARH- 

GAP22 

Akt1 human S16 S16 ARRARSKsLVMGEQS 21969604 A Rho GTPase activator that inhibits 

Rac1; phosphorylation allows 14-3-3 

binding and regulation of cell motility 

AS160 Akt1 human T642 T642 QFRRRAHtFsHPPss 16880201, 

11994271, 

16935857 

ASK1 

Akt1, 

Akt2 

human S83 S83 ATRGRGssVGGGSRR 11154276, 

15782121, 

15911620, 

14500571, 

12697749 

ataxin-1 Akt1 human S775 S775 ATRKRRWsAPESRKL 17540008, 

12757707 

B-Raf 

Akt1, 

Akt3 

human S365, 

S429 

S365, 

S429 

GQRDRsssAPNVHIN, 

PQRERKsssSsEDRN 

10869359, 

18451171 

BAD Akt1 human S99 S99 PFRGRsRsAPPNLWA 11020382, 

10558990, 

19667065 

Akt1 mouse S112, 

S155 

Bcl-10 Akt1 human S218, 

S231 

S75, 

S118 

S218, 

S231 

ETRsRHssyPAGtEE, 

GRELRRMsDEFEGSF 

EEGTCANsSEMFLPL, 

PLRSRtVsRQ_____ 

Insulin stimulated Rab GTPase-activating 

protein, structurally and functionally 

similar to TBC1D1; phosphorylation 

results in increased Glut4 translocation 

MAPKKK, induces apoptosis via JNK 

pathway; phosphorylation inhibits activity 

and promotes survival 

14-3-3 binds to and stabilizes 

ataxin-1, which forms polyglutamine 

aggregates and neurodegeneration; 

phosphorylation promotes 14-3-3 

binding 

Signaling intermediate in Erk1/2 pathway; 

phosphorylation causes inhibition 

Pro-apoptotic protein; phosphorylation 

inhibits function and promotes survival 

9381178, 

11723239, 

10983986, 

15123689, 

10949026 

16280327 A CARD (caspase recruitment 

domain) containing protein shown to 

induce apoptosis and activate NF-κB; 

phosphorylation induces nuclear 

translocation 

Bcl-xL Akt1 rat S106 S106 LRYRRAFsDLTSQLH 18951975 Prevents apoptosis through binding 

to apoptotic proteins; phosphorylation 

promotes VDAC binding 

Bex1 Akt1 rat S105 S102 KLRERQLsHSLRAVS 16498402 A neurotrophin and nerve growth factor 

signaling adaptor molecule involved 

in promoting cell cycle progression; 

phosphorylation prevents degradation 

by the proteasome 

Bim Akt1 human S87 S87 FIFMRRssLLSRSss 16282323 Pro-apoptotic protein; phosphorylation 

promotes 14-3-3 binding/inactivation 

and cell survival 

BRCA1 Akt1 human S694, 

T509 

BRF1 Akt1 human S92, 

S203 

S694, 

T509 

S92, 

S203 

QTSKRHDsDTFPELK, 

LKRKRRPtsGLHPED 

RFRDRsFsEGGERLL, 

PRLQHsFsFAGFPSA 

20085797, 

10542266 

17030608, 

15538381 

Breast cancer susceptibility gene product, 

tumor suppressor; phosphorylation 

alters function, perhaps by preventing 

nuclear localization 

A CCCH zinc-finger protein that binds 

to AU-rich elements (ARE) found in the 

3'-untranslated regions of mRNAs and 

promotes de-adenylation and rapid 

degradation by the exosome; phosphorylation 

results in binding by 14-3- 

3 protein and inactivation of BRF1 

CACNB2 Akt1 rat S625 S630 KQRSRHKsKDRYCDK 15311280 Voltage-dependent calcium channel; 

phosphorylation regulates channel 

trafficking to plasma membrane 

CaRHSP1 Akt1 human S52 S52 tRRtRtFsAtVRASQ 15910284 RNA binding protein; phosphorylation 

effect currently unknown 

Substrate 

AMPKA 

Isoform 





Casp9 Akt1 human S196 S196 KLRRRFssLHFMVEV 9812896 Protease, initiates apoptosis; phosphorylation 

inhibits protease activity 

CBP Akt1 mouse T1872 T1871 LMRRRMAtMNTRNVP 17166829 Acetylates histone and non-histone 

proteins; phosphorylation increases 

activity 

CBY1 

Akt1, 

Akt2 

human S20 S20 TPPRKSAsLSNLHsL 18573912 An inhibitor of the Wnt signaling pathway; 

phosphorylation allows 14-3-3 

binding and β-catenin sequestration in 

the cytoplasm 

CCT2 Akt1 human S260 S260 GsRVRVDstAkVAEI 19332537 Member of the protein chaperone 

complex; effect of phosphorylation 

currently unknown 

CD34 Akt2 mouse S343 S346 yssGPGAsPETQGKA 21499536 A type I transmembrane glycophosphoprotein 

expressed by hematopoietic 

stem/progenitor cells, vascular 

endothelium and some fibroblasts as 

a negative regulator of cell adhesion; 

effect of phosphorylation currently 

unknown 

Cdc25B Akt1 mouse S351 S353 VQSKRRKsVtPLEEQ 17554083 Protein phosphatase responsible 

for cdc2 activation; phosphorylation 

promotes activation of M-phase 

promoting factor 

CDK2 Akt1 human T39 T39 LkKIRLDtETEGVPs 18354084 Cyclin-dependent kinase functioning 

in S-phase; phosphorylation increases 

cyclin A binding 

CELF1 Akt1 human S28 S28 GQVPRTWsEKDLREL 18570922 RNA-binding protein; phosphorylation 

enhances interaction with cyclin D1 

mRNA 

CENTB1 Akt1 human S554 S554 SIRPRPGsLRSKPEP 16256741 GTPase-activating protein (GAP) for 

ARF proteins; phosphorylation prevents 

recycling of b1-integrin containing 

endosomes and cell migration 

CENTG1 Akt1 human S985 S985 THLSRVRsLDLDDWP 19176382 A GTPase activating protein for ARF1 

and ARF5; phosphorylation enhances 

CENTG1 GTP binding and NF-κB 

activity 

CFLAR Akt1 human S273 S273 LLRDTFTsLGYEVQK 19339247 A regulator of apoptosis; phosphorylation 

targets CFLAR for degradation 

Chk1 Akt1 human S280 S280 AKRPRVtsGGVsEsP 15107605, 

12062056 

DNA damage effector that regulates 

G2/M transition during DNA damage; 

phosphorylation inhibits function by 

preventing phosphorylation by ATM/ATR 

CK1-D Akt1 rat S370 S370 MERERKVsMRLHRGA 17594292 Kinase and core component of circadian 

clock; phosphorylation inhibits 

kinase activity 

CLK2 Akt1 human S34, 

T127 

S34, 

T127 

HKRRRSRsWSSSSDR, 

RRRRRSRtFSRSSSQ 

20682768 A dual specificity serine/threonine and 

tyrosine kinase; phosphorylation 

increases cell survival after ionizing 

radiation 

Cot Akt1 human S400 S400 EDQPRCQsLDSALLE 12138205 Oncogene; phosphorylation induces 

NF-κB-dependent transcription 

CREB Akt1 rat S133 S133 EILsRRPsYRkILND 9829964 bZIP transcription factor that activates 

target genes through cAMP response 

elements; activated by phosphorylation 

CTNNB1 

Akt1, 

Akt2 

human S552 S552 QDtQRRtsMGGtQQQ 17287208 Wnt signaling pathway protein; phosphorylation 

causes nuclear localization 

CTNND2 Akt1 mouse T454 T457 tGTFRtstAPssPGV 17993462 Transcriptional activator, plays a role 

in adhesion molecule regulation; 

phosphorylation promotes binding to 

p190RhoGEF, dendritic morphogenesis 

Cx43 Akt1 rat S369 S369 RPssRAssRAssRPR 18163231 Gap junction protein; phosphorylation 

Akt1, 

Akt3 

rat S373 S373 RAssRAssRPRPDDL 17008717, 

18163231 

allows 14-3-3 binding 

DLC1 Akt1 rat S330 S766 VTRTRSLsTCNKRVG 16338927 Tumor suppressor and insulin stimulated 

phosphoprotein, may play role in 

Glut4 translocation; phosphorylation 

may inhibit its GAP activity 

DNAJC5 Akt1 rat S10 S10 DQRQRsLsTSGESLY 16243840 Exocytosis; phosphorylation regulates 

the kinetics of late stage exocytosis 

Akt Signaling 

Research 

Please visit our website for additional 

resources and products relating to the 

study of Akt Signaling. 



www.cellsignal.com/csttables 

57



Substrate 

AMPKA 

Isoform 





DNMT1 Akt1 human S143 S143 RtPRRsksDGEAkPE 21151116 A maintenance methyltransferase, 

transferring proper methylation 

patterns to newly synthesized DNA 

during replication; phosphorylation 

increases DNMT1 stability and prevents 

methylation 

EDC3 

Akt1, 

Akt2 

human S161 S161 sFRRRHNsWssSsRH 20051463 Involved in removal of the mRNA 5’ cap 

structure; phosphorylation induces 14- 

3-3 protein interaction and promotes 

ED3 mediated post-transcriptional 

regulation through mRNA 

EDG-1 Akt1 human T236 T236 RTRSRRLtFRKNISK 11583630 G protein-coupled receptor; phosphorylation 

activates signaling to promote 

cell migration 

eIF4B Akt1 mouse S422 S422 RERsRtGsEssQtGA 18836482 Necessary for binding of mRNA to 

ribosomes; phosphorylation increases 

transcriptional activity 

ENaC-a Akt1 rat S621 S594 RFRSRYWsPGRGARG 21220922 An amiloride sensitive epithelial sodium 

channel that mediates sodium reabsorption; 

phosphorylation increases 

ENaC specific activity 

eNOS Akt1 human S615, 

S1177 

S615, 

S1177 

SYKIRFNsISCSDPL, 

TsRIRtQsFsLQERQ 

12511559, 

12446767, 

10376603, 

18622039, 

12171920 

Enzyme that catalyzes the production 

of nitric oxide (NO); phosphorylation 

results in enzyme activation, 

NO production, and cardiovascular 

homeostasis (vasodilation, vascular 

remodeling, angiogenesis) 

EphA2 Akt1 human S897 S897 RVsIRLPstsGsEGV 19573808 Receptor tyrosine kinase that binds 

to a GPI-anchored ephrin A ligand for 

regulation of cell adhesion, cell migration, 

axon guidance, and homeostasis; 

phosphorylation regulates EphA2 

induced cell migration and invasion 

ER-a 

Akt1, 

Akt2 

human S167 S167 GGRERLAsTNDKGSM 11139588, 

16113102, 

11507039 

Akt1 human S305 S305 IkRSkkNsLALSLtA 20101208 

Nuclear receptor and transcription 

factor; phosphorylation activates the 

receptor and increases gene expression, 

causing mammary and uterine 

cell proliferation 

ER-b Akt1 mouse S236 D236 VRRQRSAsEQVHCLN 17166829 Nuclear receptor and transcription factor; 

phosphorylation prevents cofactor 

binding and decreases activity 

EZH2 Akt1 human S21 S21 CWRKRVKsEYMRLRQ 16224021 Methyltransferase; phosphorylation 

decreases histone H3 methylation of 

Lys27 and increases gene expression 

ezrin Akt2 human T567 T567 QGRDKYKtLRQIRQG 15531580 Plasma membrane/cytoskeletal linker 

protein; phosphorylation promotes actin 

binding and cytoskeletal organization 

FANCA Akt1 human S1149 S1149 CLRSRDPsLMVDFIL 11855836 ATPase involved in DNA repair; 

phosphorylation is negatively regulated 

by Akt 

FLEG1 Akt1 human S486 S486 GLEtRRLsLPSsKAK 17256767 A chaperone protein involved in directing 

specific histones to the centromere; 

phosphorylation allows binding to 

14-3-3 

FLNC 

Akt1, 

Akt2 

human S2233 S2233 LGRERLGsFGsItRQ 15461588 Muscle-specific filamin functioning in 

muscle cells; phosphorylation effect 

currently unknown 

FOXA2 Akt1 human T156 T156 KTYRRSYtHAKPPYS 14500912 Transcription factor involved in embryonic 

development and differentiation; 

phosphorylation results in nuclear 

exclusion and inhibition of FoxA2- 

dependent transcriptional activity 

FOXG1 Akt1 human T279 T279 KLRRRSttsRAKLAF 17435750 Transcriptional repression factor 

involved in brain development; phosphorylation 

promotes nuclear export 

FOXO1A Akt1 human S256, 

S319, 

T24 

S256, 

S319, 

T24 

sPrRrAAsMDNNSkF, 

TFRPRtssNAsTIsG, 

LPRPRSCtWPLPRPE 

15668399, 

10358075, 

11237865, 

16076959, 

11311120 

Transcription factor involved in cell cycle 

arrest, apoptosis, and glucose metabolism; 

phosphorylation causes export 

from the nucleus and inhibits activity 

AMPKA Published Data 

Substrate Isoform 

FOXO3A Akt1 human S253, 

T32 

FOXO4 Akt1 human S197, 

S262, 

T32 

Human 


S253, 

T32 

S197, 

S262, 

T32 

APRRRAVsMDNSNKY, 

QSRPRsCtWPLQRPE 

APRRRAAsMDSSSKL, 

TFRPRSssNASSVST, 

QSRPRsCtWPLPRPE 

10910908, 

10995739, 

10102273, 

11154281 

11313479, 

11313479, 

10217147, 

16272144 



Transcription factor involved in cell 

cycle arrest and apoptosis; phosphorylation 

causes export from the nucleus 

and inhibits activity 

Transcription factor involved in cell 

cycle arrest, apoptosis, and insulin signaling; 

phosphorylation causes export 

from the nucleus and inhibits activity 

Gab2 Akt1 human S159 S159 LLRERKSsAPSHsSQ 11782427 Docking/scaffolding protein, protooncogene, 

RTK signaling intermediate; 

phosphorylation inhibits activity 

GABRB2 Akt1 rat S434 S434 SRLRRRAsQLKITIP 12818177 Receptor that mediates fast inhibitory 

synaptic transmission in the brain; 

phosphorylation increases the number 

of receptors on the cell surface 

GAPDH Akt2 human T237 T237 GMAFRVPtANVSVVD 21979951 Catalyzes the phosphorylation of 

glyceraldehyde-3-phosphate during 

glycolysis; phosphorylation decreases 

nuclear translocation and GAPDH 

induced apoptosis 

GATA1 Akt1 human S310 S310 QTRNRKAsGkGkkkR 16107690 Transcription factor; phosphorylation 

increases activity and promotes blood 

cell differentiation 

GATA2 Akt1 human S401 S401 QTRNRKMsNKSKKSK 15837948 Transcription factor; phosphorylation 

inhibits activity to promote adipogenesis 

and reduce inflammation 

girdin Akt1 human S1417 S1417 INRERQKsLtLTPTR 16139227 Actin binding protein; phosphorylation 

promotes cell migration 

GOLGA3 Akt1 mouse S174, 

S385 

S174, 

S389 

VKRHRERsSQPAtKM, 

EVRsRRDsICsSVSM 

17888676 Golgi auto-antigen; phosphorylation 

results in reduced apoptosis 

Grb10 Akt1 mouse S455 S428 NAPMRsVsENsLVAM 15722337 An adaptor protein that interacts with 

many receptor tyrosine kinases as well 

as downstream signal molecules; phosphorylation 

allows binding to 14-3-3 

GSK-3a Akt1 human S21 S21 SGRARtssFAEPGGG 11340086, 

11563975, 

11577096 

GSK-3b Akt1 human S9 S9 SGRPRttsFAESCKP 12900420, 

15457186, 

11563975, 

11340086, 

11577096, 

8985174 

Serine/threonine protein kinase 

that phosphorylates and inactivates 

glycogen synthase; phosphorylation 

inhibits activity 

Serine/threonine protein kinase 

that phosphorylates and inactivates 

glycogen synthase; phosphorylation 

inhibits activity 

H2B Akt1 human S37 S37 RKRsRkEsysIyVyk 8985174 Core component of the nucleosome; 

phosphorylation effect currently 

unknown 

H3 Akt1 mouse S10 S10 tKQTARksTGGkAPR 12529330 Core component of the nucleosome; 

phosphorylation is correlated with 

chromosome condensation during 

mitosis and meiosis 

HMOX1 Akt1 human S188 S188 LYRSRMNsLEMtPAV 15581622 Heme oxygenase involved in stress 

response; phosphorylation regulates 

binding affinity 

hnRNP A1 Akt1 human S199 S199 sQrGrsGsGNFGGGr 18562319 Involved in pre-mRNA packaging 

into hnRNP particles and transport 

of poly(A) mRNA from cytoplasm to 

nucleus; phosphorylation regulates role 

in cyclin D1 and c-Myc IRES activity 

hnRNP E1 Akt1, 

Akt2 

mouse S43 S43 VKRIREEsGARINIS 20154680 Binds to single-stranded nucleic acid; 

phosphorylation results in disruption of 

BAT element binding and translational 

activation of Dab2 and ILEI mRNA 

HSP27 Akt1 human S82 S82 RALsRQLssGVSEIR 12740362 Heat shock protein that confers cellular 

resistance to stress and adverse 

environmental change; phosphorylation 

alters tertiary structure, modulates 

actin polymerization, and reorganization 

HTRA2 

Akt1, 

Akt2 

human S212 S212 RVRVRLLsGDTYEAV 17311912 Protease released during apoptosis; 

phosphorylation inhibits activity and 

attenuates its pro-apoptotic function 





Substrate 

Huntingtin 

IKK-a 

AMPKA 

Isoform 



Akt1 human S421 S421 GGRsRsGsIVELIAG 12062094, 

14725621, 

15843398, 

16452687 

Akt1, 

Akt2 

human T23 T23 EMRERLGtGGFGNVC 18515365, 

12048203, 

10485710, 

19609947 



Huntington’s disease; Akt phosphorylation 

blocks nuclear aggregation and 

provides neuroprotection 

NF-κB signaling intermediate; 

phosphorylation activates NF-κB and 

immune/stress response 

IP3R1 Akt1 rat S2682 S2690 FPRMRAMsLVSSDSE 16332683 Ca 2+ release and signaling; phosphorylation 

induces resistance to apoptosis, 

possibly through caspase-3 inactivation 

IRAK1 Akt1 human T100 T100 LRARDIItAWHPPAP 11976320 A serine/threonine-specific IL-1 

receptor-associated kinase involved in 

Toll signaling; phosphorylation inhibits 

IRAK mediated NK-κB activation 

IRS1 Akt1 human S629 S629 VPSGRKGsGDyMPMs 17640984 Insulin receptor signaling intermediate; 

Akt1 rat S522 S527 RFRKRTHsAGTSPTI 17579213 

phosphorylation inhibits function 

KHSRP 

Kv11.1 

iso5 

Lamin 

A/C 

Akt1, 

Akt2 

human S193 S193 GLPERSVsLTGAPES 17177604 Recruits degradation machinery, 

activates mRNA turnover, regulates 

splicing; phosphorylation inhibits RNA 

turnover by degradation 

Akt1 human T897 T897 SFRRRtDtDtEQPGE 18791070 Pore-forming subunit of voltage-gated 

potassium channels, essential for 

rhythmic excitability of cardiac muscle 

and endocrine cells; phosphorylation 

inhibits channels 

Akt1 rat S301, 

S404 

S301, 

S404 

RSRGRASsHSSQSQG 18808171 Component of nuclear lamina; 

phosphorylation regulates function of 

nuclear lamina 

LTB4R2 Akt1 human T355 T355 GGRsREGtMELRTTP 22044535 A low-affinity leukotriene receptor 

involved in chemotaxis; phosphorylation 

regulates activation of chemotactic 

responses 

Mad1 Akt1 human S145 S145 IERIRMDsIGSTVSS 18451027, 

19526459 

MDM2 Akt1 human S166, 

S186, 

S188 

S166, 

S186, 

S188 

SsRRRAIsEtEENsD, 

RQRKRHKsDsIsLsF, 

RKRHKsDsIsLsFDE 

11715018, 

15169778, 

11504915, 

11850850, 

11923280, 

15527798, 

11960368 

Component of spindle-assembly 

checkpoint; phosphorylation results in 

ubiquitination and degradation through 

26S proteasome pathway 

Ubiquitin ligase involved in p53 

degradation; phosphorylation results 

in translocation to the nucleus and 

inhibition of p53 

MDM4 Akt1 human S367 S367 PDCRRtIsAPVVRPK 18356162 RING-finger domain protein involved 

in p53 degradation and apoptosis; 

phosphorylation stabilizes MDM4 and 

MDM2 

METTL1 Akt1 human S27 S27 yYRQrAHsNPMADHT 15861136 Catalyzes the formation of m7G46 

in tRNA; phosphorylation results in 

inactivation 

MKK4 Akt1 human S80 S80 IERLRtHsIEsSGKL 15911620, 

11707464 

Signaling intermediate of the JNK/ 

SAPK pathway involved in stress/ 

inflammation; phosphorylation inhibits 

activity 

MLK3 Akt1 human S674 S674 PGRERGEsPTtPPTP 12458207 JNK-mediated neuronal cell death; 


MST1 Akt1 human T120 T120 IIRLRNktLTEDEIA 19940129 Pro-apoptotic kinase; phosphorylation 

inhibits kinase activity and nuclear 

translocation resulting in inhibition of 

pro-apoptotic signaling 

MST2 Akt1 human T117, 

T384 

mTOR Akt1 human T2446, 

S2448 

T117, 

T384 

T2446, 

S2448 

IIRLRNktLIEDEIA, 

GTMKRNAtsPQVQRP 

20231902, 

20086174 

RsRtRtDsysAGQsV 15208671, 

10910062, 

10567225 

Upstream activator of the MAPK 

pathway that regulates apoptosis, 

morphogenesis, and cytoskeletal rearrangements; 

phosphorylation inhibits 

pro-apoptotic activity 

Protein synthesis and cell growth; 

phosphorylation increases activity 

Substrate 

AMPKA 

Isoform 





MYO5A Akt2 mouse S1650 S1652 GLRKRtssIADEGty 17515613 Actin-based motor protein with a role 

in cytoplasmic vesicle transport and 

anchorage; phosphorylation promotes 

insulin-mediated Glut4 vesicle 

translocation 

Myt1 Akt1 starfish S75 S83 ESRPRAVsFRQSEPS 11802161 Wee1 family member and cell cycle 

regulator; phosphorylation downregulates 

Myt1 and initiates M-phase 

NDRG2 Akt1 human S332, 

T348 

S332, 

T348 

LsRsRtAsLtsAAsV, 

GNRsRsRtLsQssEs 

NFAT90 Akt1 human S647 S647 rGrGRGGsIRGRGRG 18097023, 

20870937 

NHE1 Akt1 human S648, 

S703, 

S796 

S648, 

S703, 

S796 

KTRQRLRsyNRHTLV, 

MsRARIGsDPLAyEP, 

QRIQRCLsDPGPHPE 

15461589 Insulin-stimulated phosphoprotein; 

phosphorylation promotes insulin 

signaling 

18757828, 

20026127 

Translation inhibitory protein; phosphorylation 

required for nuclear export 

Sodium/hydrogen exchanger involved 

in pH regulation and signal transduction; 


NMDAR2C Akt1 mouse S1084 S1081 GPRPRHAsLPSSVAE 19477150 Glutamate receptor channel subunit; 

Akt1 rat S1083 S1081 GPRPRHAsLPSSVAE 19477150 

phosphorylation promotes binding 

to 14-3-3ε and leads to increased 

surface expression of cerebellar NMDA 

receptors 

NuaK1 Akt1 human S600 S600 PARQRIRsCVSAENF 15060171, 

12409306 

Nur77 Akt1 human S351 S351 GRRGRLPsKPKQPPD 16434970, 

11274386 

p21 Cip1 Akt1 human S146, 

T145 

p27Kip1 Akt1 human S10, 

T157, 

T198 

S146, 

T145 

S10, 

T157, 

T198 

GRkRRQtsMTDFYHs, 

QGRkRRQtsMTDFYH 

NVRVsNGsPsLErMD, 

GIRkrPAtDDSSTQN, 

PGLRRRQt_______ 

AMPK family member activated under 

glucose starvation that mediates cell 

survival; phosphorylation increases 

kinase activity 

A nuclear receptor and transcription 

factor regulating T cell apoptosis; 

phosphorylation inhibits transcriptional 

activity 

17855660, Regulates cell cycle and cell survival; 

11231573, phosphorylation increases protein 

11756412, stability 

15173090, 

11463845, 

116982699 

18710949, 

12042314, 

12244302 

p300 Akt1 human S1834 S1834 MLRRRMAsMQRTGVV 16024795, 

11116148 

p47phox Akt1 human S304, 

S328 

S304, 

S328 

GAPPRRssIRNAHSI, 

QDAYRRNsVRFLQQR 

A cyclin-dependent kinase inhibitor that 

enforces the G1 cell cycle restriction 

point; phosphorylation promotes 14-3- 

3 binding and cytoplasmic localization 

Transcriptional co-activator; phosphorylation 

can either activate or suppress 

transcriptional activity depending on 

cell type and physiological stimuli 

12734380 A component of the phagocytic NADPH 

oxidase multiprotein enzyme that 

catalyzes the reduction of oxygen to 

superoxide in response to pathogenic 

invasion; phosphorylation regulates 

p47hox respiratory burst activity 

PAK1 Akt1 mouse S21 S21 APPMRNTsTMIGAGS 14585966 A p21-activated kinase engaged in 

cytoskeletal reorganization, MAPK signaling, 

apoptotic signaling, control of 

phagocyte NADPH oxidase, and growth 

factor-induced neurite outgrowth; 

phosphorylation at Ser21 regulates 

binding with the adaptor protein Nck 

palladin Akt1 human S1118 S1118 VRRPRsRsRDsGDEN 20471940 Actin-bundling protein; phosphorylation 

promotes F-actin bundling and inhibits 


PAR-4 Akt1 rat S249 N257 SRHNRDTsAPANFAS 16209943 A pro-apoptotic factor that activates 

the Fas-FADD-caspase-8 pathway as 

well as inhibits the NF-κB pro-survival 

pathway; phosphorylation prevents 

nuclear translocation, promoting cell 

survival 

PDCD4 Akt1 human S67, 

S457 

PDE3A Akt1 mouse S290, 

S291, 

S292 

S67, 

S457 

S290, 

S291, 

S292 

kRRLRKNssRDsGRG, 

RGRKRFVsEGDGGRL 

GWKRRRRsssVVAGE, 

WKRRRRsssVVAGEM, 

KRRRRsssVVAGEMS 

16357133 Tumor suppressor protein that is 

strongly induced during apoptosis; 

phosphorylation inhibits tumor suppressor 

function 

17124499 Regulates levels of cAMP and cGMP, 

insulin-dependent oocyte maturation; 


Akt Signaling 

Research 







61



Substrate 

AMPKA 

Isoform 





PDE3B Akt1 mouse S273 S295 VIRPRRRssCVsLGE 10454575 Regulates levels of cAMP and cGMP, 

activated by insulin to regulate lipolysis; 


PEA-15 Akt1 human S116 S116 KDIIRQPsEEEIIKL 12808093 A phosphoprotein shown to coordinate 

cell growth, death, and glucose utilization; 

phosphorylation mediates binding 

to FADD or Erk and further regulates 

the Erk and apoptosis signaling 

pathways 

peripherin Akt1 mouse S66 S59 SSSARLGsFRAPRAG 17569669 Neuronal intermediate filament protein; 

phosphorylation promotes motor nerve 

regeneration 

PFKFB2 Akt1 human S466, 

S483 

S466, 

S483 

PVRMRRNsFtPLSSS, 

IRRPRNysVGSRPLK 

12853467 Glycolytic enzyme, insulin-mediated 

glucose metabolism; phosphorylation 

increases activity 

PFKFB3 Akt1 human S461 S461 NPLMRRNsVtPLAsP 15896703 Synthesis and degradation of fructose 

2,6-bisphosphate; phosphorylation 

decreases sensitivity to inhibition 

PGC-1 a 

Akt1, 

Akt2 

mouse S570 S571 RMRSRsRsFsRHRSC 17554339 Regulates gluconeogenesis and fatty 

acid oxidation; phosphorylation inhibits 

function 

PIP5K Akt1 human S307 S307 PARNRsAsItNLsLD 15546921 A protein/ lipid kinase involved in 

Akt1 mouse S105 S105 EELHRRSsVLENTLP 20513353 

membrane trafficking; phosphorylated 

in response to insulin 

PLB Akt1 rat S16 S16 RSAIRRAstIEMPQQ 18838385 A major phosphoprotein calcium regulation 

component of the sarcoplasmic 

reticulum; phosphorylation causes 

release of inhibition and increases 

calcium uptake by the sarcoplasmic 

reticulum 

PLCG1 Akt1 human S1248 S1248 HGRAREGsFEsRyQQ 16525023 Catalyzes PI 4,5 bisphosphate to IP 3 

and DAG, increases intracellular Ca 2+ 

levels; phosphorylation increases 

activity and enhances EGF-stimulated 

cell motility 

PPP1CA Akt1 human T320 T320 NPGGRPItPPRNSAK 14633703 A serine/threonine phosphatase 

involved in cell cycle regulation; 


PRAS40 Akt1 human T246 T246 LPRPRLNtsDFQKLK 12524439, 

17277771, 

18372248 

Binds to and inhibits mTOR; phosphorylation 

causes 14-3-3 binding/ 

inhibition and results in increased 

protein synthesis 

PRPF19 Akt1 human T193 T193 ERKKRGKtVPEELVK 20629186 A member of the splicesome that also 

functions in DNA double strand break 

repair; phosphorylation allows 14-3-3 

binding 

PRPK Akt1 human S250 S250 RLRGRKRsMVG____ 17712528 p53 binding protein and kinase; 

phosphorylation causes activation and 

results in p53 phosphorylation 

PTP1B Akt1 human S50 S50 RNRyRDVsPFDHsRI 11579209 Protein tyrosine phosphatase that 

dephosphorylates the insulin receptor; 


QIK Akt2 mouse S358 S358 DGRQRRPstIAEQTV 17805301 AMPK related protein; phosphorylation 

leads to kinase activation and promotes 

ubiquitination/degradation of TORC2 

Rac1 Akt1 human S71 S71 yDRLRPLsYPQTDVF 10617634 Rho-GTPase, actin cytoskeletal 

organization; phosphorylation inhibits 

GTP-binding activity 

Raf1 Akt1 mouse S259 S259 SQRQRStsTPNVHMV 12087097, 

12087097 

Akt1 rat S259 S259 SQRQRSTsTPNVHMV 11443134 

Signaling intermediate in Erk1/2 pathway; 


RANBP3 Akt1 human S126 S126 VKRERtssLtQFPPs 18280241 RAN binding protein 3 functions in 

nuclear transport; phosphorylation 

mediates Ran binding and regulates 

nuclear transport 

RARA Akt1 human S96 S96 FVCQDKSsGYHYGVS 16417524 Nuclear receptor for retinoic acid that 

acts as a direct regulator of gene 

expression, phosphorylation of the DNA 

binding domain inhibits RARA activity 

Substrate 

AMPKA 

Isoform 





RGC32 Akt1 human S65 S65 ERMKRRSsAsVSDSS 19162005 A regulator of cell cycle-specific 

kinases in response to DNA damage; 

phosphorylation leads to activation and 

regulation of growth factors 

RNF11 Akt1 human T135 T135 DWLMRSFtCPSCMEP 16123141 A member of a ubiquitin editing 

complex that modulates transient 

inflammatory signaling; phosphorylation 

allows 14-3-3 binding 

Ron Akt1 human S1394 S1394 VRRPRPLsEPPRPT_ 12919677, 

14505491 

Receptor tyrosine kinase for macrophage 

stimulating protein (MSP), cell 

adhesion, proliferation and migration; 

phosphorylation causes 14-3-3 binding 

RPS3 Akt1 human T70 T70 GrrIrELtAVVQkRF 20605787 A member of the 40S ribosomal subunit 

that also induces neuronal apoptosis 

and acts as an endonuclease; 

phosphorylation inhibits proapoptotic 

function, increases nuclear import/accumulation, 

and increases DNA repair 

S6 Akt1 mouse S236 S236 AKRRRLssLRAstsK 12151408 S6 ribosomal protein; phosphorylation 

Akt1, rat S235, S235, IAKRRRLssLRAsts, 15358595 

activates the protein and promotes 

protein synthesis 

Akt2 

S236 S236 AKRRRLssLRAstsK 

SFRS5 Akt2 rat S86 S86 GRGRGRYsDRFSSRR 15684423 A member of the splicesome involved 

in constitutive and alternative splicing; 

phosphorylation activates alternative 

splicing exon inclusion 

SH3BP4 Akt1 mouse S245 S246 FRSKRSysLsELsVL 19122209 Controls selective internalization of the 

transferrin receptor through endocytosis; 

phosphorylation promotes 14-3-3 

binding at the plasma membrane 

SH3RF1 

Akt1, 

Akt2 

human S304 S304 KNTKKRHsFtsLTMA 17535800 Scaffolding protein that binds to 

activated Rac and promotes apoptosis 

via JNK activation; phosphorylation 

reduces ability to bind Rac, promoting 

apoptosis 

SKI Akt1 human T458 T458 QPRKRKLtVDTPGAP 19875456 Negative regulator of TGF-b signaling 

by binding to Smads; phosphorylation 

causes its destabilization and reduces 

SKI-mediated inhibition of expression 

of Smad7 

SOX2 Akt1 mouse T118 T116 kYRPRRktkTLMkKD 20945330 A transcription factor required for early 

embryogenesis and embryonic stem 

cell pluripotency; phosphorylation 

stabilizes SOX2, increasing transcriptional 

activity 

SRPK2 Akt1 human T492 T492 PSHDRSRtVsAsstG 19592491 A protein kinase targeting the serine/ 

arginine family of splicing factors; 

phosphorylation causes nuclear translocation 

and upregulation of targets 

regulating cell cycle progression and 

apoptosis 

SSB Akt1 mouse T301 T302 LLRNKKVtWKVLEGH 18836485 RNA binding protein, plays a role in 

processing of RNA polymerase III 

transcripts; phosphorylation promotes 

export to cytoplasm where it binds 

polysomes and regulates expression of 

a specific set of mRNAs 

STXBP4 Akt2 mouse S99 S99 RAKLRsEsPWEIAFI 15753124 Inhibits formation and translocation of 

intracellular vesicles; insulin-stimulated 

phosphorylation of STXBP4 releases 

inhibition 

SYTL1 Akt1 human S241 S241 RMLSSSSsVSSLNSS 15998322 A secretory factor family member that 

is involved in granule exocytosis; phosphorylation 

regulates SYTL1 subnuclear 

localization 

TAL1 Akt1 human T90 T90 EARHRVPttELCRPP 15930267, 

19406989 

Transcription factor; phosphorylation 

inhibits transcriptional repressor activity 

and regulates intracellular localization 

TBC1D1 Akt1 human T596 T596 AFRRRANtLsHFPIE 17995453 Rab GTPase-activating protein involved 

in insulin-stimulated Glut4 trafficking; 

phosphorylation promotes glucose 

transport 







TERT Akt1 human S227, 

S824 

THOC4 Akt1 human S34, 

T219 

Human 


S227, 

S824 

S34, 

T219 

GARRRGGsASRSLPL, 

AVRIRGKsYVQCQGI 

RGRGRAGsQGGrGGG, 

GGGtrRGtRGGARGR 



10224060 Telomerase reverse transcriptase, 

chromosome length maintenance; 

phosphorylation enhances telomerase 

activity 

18562279 An RNA binding and export protein 

that also acts as a chaperone for 

dimerization of transcription factors; 

phosphorylation regulates THOC4 

subnuclear localization and activates 

mRNA export and cell proliferation 

TOPBP1 Akt1 human S1159 S1159 EERARLAsNLQWPSC 19477925 Induces a large increase in the kinase 

activity of ATR; phosphorylation prevents 

the enhanced association of ATR 

with TopBP1 after DNA damage 

TRF1 Akt1 human T273 T273 SKRTRTItSQDKPSG 19160102 Controls telomere structure; phosphorylation 

decreases telomere length 

TSC2 Akt1 human S939, 

S981, 

T1462 

Akt1 rat S1130, 

S1132 

S939, 

S981, 

T1462 

S1130, 

S1132 

sFRARstsLNERPKs, 

AFRCRSIsVSEHVVR, 

GLRPRGytIsDSAPs 

GARDRVRsMsGGHGL, 

RDRVRsMsGGHGLRV 

15342917, 

12150915, 

16636147 

12172553 

Tumor suppressor that inhibits mTOR; 

phosphorylation inhibits function and 

allows protein synthesis to occur 

TTC3 Akt1 human S378 S378 AYTPRsLsAPIFTTS 20059950 E3 ligase to Akt; phosphorylation 

promotes TTC3 function, such as ability 

to ubiquitinylate and destabilize Akt 

TWIST1 Akt1 human S42, 

S123 

S42, 

S123 

GGRKRRSsRRSAGGG, 

RERQRTQsLNEAFAA 

20400976 A regulatory basic helix-loop-helix 

anti-apoptotic transcription factor; 

phosphorylation activates TWIST1, 

causing inhibition of p53 and promotion 

of cell survival 

USP8 Akt1 mouse T907 T945 TCRRRSRtFEAFMYL 17210635 Deubiquitinating enzyme that plays a 

role in growth factor receptor trafficking 

and degradation; phosphorylation 

increases protein stability 

VCP Akt1 human S352, 

S746, 

S748 

S352, 

S746, 

S748 

AAtNRPNsIDPALRR, 

AMRFARRsVsDNDIR, 

RFARRsVsDNDIRky 

16551632, 

16027165 

ATPase and molecular chaperone; 

phosphorylation may impair its proapoptotic 

effects and promote cell 

survival 

Vimentin Akt1 human S39 S39 ttsTrtysLGsALRP 20856200 A cytoskeletal intermediate filament 

protein; phosphorylation induces cellular 

motility and invasion by protection 

from proteolysis 

Wee1 Akt1 human S642 S642 KKMNRsVsLTIy___ 15964826 A protein kinase that inhibits cell cycle 

progression by phosphorylation inhibition 

of cdc2 kinase; phosphorylation 

promotes a change in Wee1 localization 

from nuclear to cytoplasmic and is 

associated with G2/M arrest 

WNK1 Akt1 human T60 T60 EYRRRRHtMDKDSRG 14611643, 

16081417 

XIAP 

Akt1, 

Akt2 

human S87 S87 VGRHRKVsPNCRFIN 14645242, 

17537996 

Regulates ion channels; phosphorylation 

of WNK1 causes SGK1 activation 

and regulation of sodium ion transport 

Inhibitor of apoptosis; phosphorylation 

prevents ubiquitination/degradation and 

causes increased cell survival 

YAP1 Akt1 human S127 S127 PQHVRAHssPAsLQL 12535517 A transcriptional co-activator of PEBP2 

and other transcription factors; phosphorylation 

suppresses p73-mediated 

apoptosis 

YB-1 Akt1 human S102 S102 NPRKyLRsVGDGEtV 22417301 A transcription/translation factor 

involved in mRNA stability and expression; 

phosphorylation induces activation 

and translocation to the nucleus 

zyxin Akt1 human S142 S142 PQPREKVssIDLEId 17572661 A focal adhesion molecule that moves 

between the cytoplasm and nucleus; 

phosphorylation promotes an association 

with acinus and anti-apoptotic 

activity 

Akt Binding Partners 

Binding 

Partners Effect of Binding 

Effect on 

Akt activity References 

GAPDH Binds to active Akt and 

limits its dephosphorylation 

Positive Jacquin, M.A. et al. (2013) Cell Death Differ. 20, 

1043–1054. 

Jade-1 Binds to and inhibits Akt kinase activity Negative Zeng, L. et al. (2013) Cancer Res. 73, 5371–5380. 

Mst1 Binds to and inhibits Akt kinase activity Negative Cinar, B. et al. (2007) EMBO J. 26, 4523–4534. • Jang, 

S.W. et al. (2007) J. Biol. Chem. 282, 30836–30844. 

ArgBP2γ Binds and functions as an 

N/A Yuan, Z.Q. et al. (2005) J. Biol. Chem. 280, 21483–21490. 

adaptor for Akt and PAK1 

CBP Akt binds and phosphorylates 

N/A Liu, Y. et al. (2013) FEBS Lett. 587, 847–853. 

CBP to regulate CBP activity 

PP1 Binds Akt and dephosphorylates Negative Xiao, L. et al. (2010) Cell Death Differ. 17, 1448–1462. 

Akt at Thr450 

PLCγ1 Akt binds and phosphorylates PLCγ1 N/A Wang, Y. et al. (2006) Mol. Biol. Cell 17, 2267–2277. 

Skp2 

PEA-15 

PHF20 

PHLPP 

FKBP5 

CKIP-1 

Ras 

BTBD10 

KCTD20 

PAR-4 

Akt binds and phosphorylates 

Skp2 to regulate Skp2 activity 

Akt binds and phosphorylates PEA-15 

to regulate its anti-apoptotic function. 

Akt binds and phosphorylates PHF20 

to regulate its subcellular localization 

PHLPP binds Akt and dephosphorylates 

Akt at Ser473 

Binds Akt and acts as a scaffold for 

the interaction of Akt with PHLPP 

Binds Akt and inhibits 

Akt phosphorylation 

Interacts with the pleckstrin homology 

domain of Akt 


its dephosphorylation 


its dephosphorylation 

Akt binds and phosphorylates PAR-4 

to inhibit its pro-apoptotic activity 

N/A Lin, H. et al. (2009) Nat. Cell Biol. 11, 420–432. • Gao, D. 

et al. (2009) Nat. Cell Biol. 11, 397–408 

N/A Trencia, A. et al. (2003) Cell. Biol. 23, 4511–4521. 

N/A Park, S. et al. (2012) J. Biol. Chem. 287, 11151–11163. 

Negative Gao, T. et al. Mol. Cell 18, 13–24. 

Negative Pei, H. et al. (2009) Cancer Cell 16, 259–266. 

Negative Tokuda, E. et al. (2007) Cancer Res. 67, 9666–9676. 

Positive Yue, Y. et al. (2004) J. Biol. Chem. 279, 12883–12889. 

Positive Nawa, M. et al. (2008) Cell Signal. 20, 493–505. 

Positive Nawa, M. et al. (2013) BMC Biochem. 14, 27. 

N/A Goswami, A. et al. (2005) Mol. Cell 20, 33–44. 

Tpl2 Akt binds and phosphorylates Tpl2 N/A Kane, L.P. et al. (2002) Mol. Cell. Biol. 22, 5962–5974. 

SirT2 SirT2 interacts with Akt and is Positive Ramakrishnan, G. et al. (2014) J. Biol. Chem. 289, 

required for optimal Akt activation 

6054–6066. 

NPM 

eEF1A 

CLIPR-59 

Binds the pleckstrin homology domain 

of Akt to promote cell survival 

Interacts with Akt and contributes 

to Akt phosphorylation 

Interacts with the kinase domain 

of Akt and regulates the subcellular 

localization of Akt 

N/A Lee, S.B. et al. (2008) Proc. Natl. Acad. Sci. USA 105, 

16584–16589. • Kwon, I.S. et al. (2010) BMB Rep. 43, 

127–132. 

Positive Pecorari, L. et al. (2009) Mol. Cancer 8, 58. 

N/A Ding, J. et al. (2009) Mol. Cell. Biol. 29, 1459–1471. 

CNK1 Binds and enhances Akt activation Positive Fritz, R.D. et al. (2010) Oncogene 29, 3575–3582. 

Phafin2 Binds Akt in the lysosome 

N/A Matsuda–Lennikov, M. et al. (2014) PLoS One 9, e79795. 

to regulate autophagy 

Btk Binds Akt and promotes 

Akt phosphorylation 

Positive Lindvall, J. et al. (2002) Biochem. Biophys. Res. Commun. 

293, 1319–1326. 

β-Parvin Binds Akt and prevents the 

interaction of Akt with ILK 

Negative Kimura, M. et al. (2010) J. Cell Sci. 123, 747–755. 

NS1 

Interacts with the pleckstrin 

homology domain of Akt 

Positive 

Matsuda, M. et al. (2010) Biochem. Biophys. Res. Commun. 

395, 312–317. 

α-Synuclein Binds Akt and promotes Akt activation Positive Chung, J.Y. et al. (2011) Neurosignals 19, 86–96. 

RACK1 RACK1 interacts with Akt 

Negative Li, G. et al. (2012) Nat. Commun. 3, 667. 

in a complex with PP2A 

ProF Binds Akt and influences 

N/A Fritzius, T. et al. (2006) Biochem. J. 399, 9–20. 

the subcellular localization of Akt 

p27 Kip1 Akt binds and phosphorylates p27 N/A Liang, J. et al. (2002) Nat. Med. 8, 1153–1160. • Shin, I. 

et al. (2002) Nat. Med. 8, 1145–1152. 

FoxA2/HNF3β Akt binds and phosphorylates 

FoxA2/HNF3β 

N/A Wolfrum, C. et al. (2003) Proc. Natl. Acad. Sci. USA 100, 

11624–11629. 

DNMT1 Akt binds and phosphorylates DNMT1 N/A Estève, P.O. et al. (2011) Nat. Struct. Mol. Biol. 18, 42–48. 

Akt Signaling 

Research 







65

FGFR2 FGFR3 

TrkC 

EphB2 

TrkB 

FGFR1 

FGFR4 

EphB1 

TrkA 

FLT1/VEGFR1 

EphA5 

KDR/VEGFR2 

MuSK ROR2 

Fms/CSFR 

ROR1 

EphB3 

Ret 

Kit 

EphA3 

DDR2 

Mer 

EphA4 

DDR1 

Tyro3/ 

Axl 

FLT4 

Sky 

PDGFRα 

EphA6 

IGF1R IRR 

FLT3 

PDGFRβ 

InsR 

Yes 

EphB4 

Met 

EGFR HER2/ErbB2 

Src 

Ron 

MLK3 

Ros 

EphA7 

ALK 

MLK1 

Lyn 

LTK 

Tie2 

Tie1 

HCK 

Fyn 

RYK 

HER4 

EphA8 

MLK4 

CCK4/PTK7 

MLK2 

Lck 

Fgr 

Ack Tnk1 Tyk2 

Jak1 

HER3 

Jak2 

EphA2 

Jak3 

BLK 

ANKRD3 SgK288 

DLK 

Syk Zap70/SRK 

EphA1 

PYK2/FAK2 

LZK 

FAK 

Lmr1 

ALK4 

ITK 

Lmr2 

C-Raf/Raf1 

FRK 

TGFβR1 

ZAK 

BRaf 

TEC 

EphB6 

KSR 

Srm 

RIPK2 

KSR2 

TXK 

Brk 

BTK 

Lmr3 

ALK7 

IRAK3 

IRAK1 

LIMK1 

ARaf 

BMPR1B 

Etk/BMX 

EphA10 

LIMK2 TESK1 ILK 

BMPR1A 

CTK 

RIPK3 

TSK2 

TAK1 

HH498 

ALK1 

CSK 

ALK2 

Abl2/Arg 

IRAK2 

ActR2 

Abl Fes 

ActR2B 

Fer 

RIPK1 

Jak3~b 

TGFβR2 

LRRK2 

MEKK2/MAP3K2 

Jak2~b 

LRRK1 

MISR2 

MEKK3/MAP3K3 

Tyk2~b 

SuRTK106 

IRAK4 

BMPR2 

ASK/MAP3K5 

ANPα/NPR1 

MAP3K8 

Jak1~b 

MAP3K7 

ANPβ/NPR2 

KHS1 

MOS 

KHS2 

HSER 

SgK496 

WNK1 

WNK3 

MEKK6/MAP3K6 

DYRK2 

Mst4 

PBK 

MAP3K4 

DYRK3 

GUCY2D 

WNK2 

DYRK4 

DYRK1A 

GUCY2F 

OSR1 

DYRK1B 

WNK4 

MLKL 

PERK/PEK 

SgK307 

SLK 

PKR 

LOK 

HIPK1 HIPK3 

GCN2 SgK424 

TAO1 

SCYL3 TAO2 

HIPK2 

SCYL1 

Tpl2/COT 

SCYL2 

NIK 

TAO3 

PAK1 

HIPK4 

PRP4 

HRI 

PAK3 PAK4 

CLIK1 

PAK2 

IRE1 

MAP2K5 

PAK5/PAK7 

CLIK1L 

IRE2 

CLK3 

TBCK 

PAK6 

MAP2K7 

MEK1/MAP2K1 

RNAseL 

GCN2~b 

MEK2/MAP2K2 

TTK 

MSSK1 

SgK071 

SRPK2 

KIS 

MYT1 

SEK1/MAP2K4 MKK3/MKK6 

SRPK1 

CK2α1 

Wee1 

MAK 

CK2α2 

SgK196 

CDC7 

Wee1B 

CK1δ 

ICK 

PRPK 

TTBK1 

CK1ε 

GSK3β 

MOK 

TTBK2 

GSK3α 

CK1α1 

CDKL3 

CK1α2 

CDKL2 

PINK1 

SgK493 

SgK269 

VRK3 

CK1γ2 

CDKL1 CDKL5 

ERK7 

SgK396 

SgK223 

CDKL4 Erk4 

Slob 

CK1γ1 

Erk3 

SgK110 

PIK3R4 

CK1γ3 

NLK 

SgK069 

Bub1 

Erk5 

SBK 

BubR1 

Erk1/ 

IKKα 

p44MAPK 

IKKβ 

VRK1 

CDK7 

IKKε 

VRK2 

Erk2/ 

PLK4 

PITSLRE 

TBK1/NAK 

p42MAPK p38γ 

MPSK1 

JNK1 

p38δ 

JNK2 

TLK2 

JNK3 CDK10 

GAK 

TLK1 

PLK3 

p38β 

AAK1 

CAMKK1 

ULK3 

PLK1 

p38α 

PLK2 

CDK4 

CCRK 

BIKE 

CAMKK2 

BARK1/GRK2 

CDK6 

ULK1 

BARK2/GRK3 RHOK/GRK1 

Fused 

GRK5 

PFTAIRE2 

SgK494 

ULK2 ULK4 

GRK6 GRK4 

PFTAIRE1 

CDK9 

Nek6 

RSKL1 

PCTAIRE2 

Nek7 Nek10 

SgK495 

PASK 

PDK1 

RSKL2 

Nek8 

MSK1 

RSK1/p90RSK 

PCTAIRE1 

CDK5 CRK7 

PCTAIRE3 

Nek9 

LKB1 

MSK2 

RSK4 RSK2 

Chk1 

p70S6K 

RSK3 

Nek2 

Akt2/PKBβ 

AurA/Aur2 

p70S6Kβ 

Akt1/PKBα 

cdc2/CDK1 

Nek11 

Akt3/PKBγ 

CDK3 

Nek4 

AurB/Aur1 

SGK1 

CDK2 

AurC/Aur3 

SGK2 

SGK3 

Trb3 Pim1 Pim2 

PKG2 

PKN1/PRK1 

LATS1 

Nek3 

PKG1 

PKN2/PRK2 

Trb2 

Pim3 

LATS2 

PKN3 

Nek5 

NDR1 

PRKY 

PKCδ 

Trb1 

Obscn~b 

NDR2 

PKCθ 

PRKX 

YANK1 

SPEG~b 

MAST3 

PKCη 

Nek1 

MASTL 

PKCε 

Obscn 

STK33 

YANK2 

PKCι 

YANK3 

PKCζ 

SPEG 

PKAγ 

PKAα 

PKCγ 

TTN 

MAST2 

PKAβ 

smMLCK 

TSSK4 

ROCK1 

PKCα 

HUNK 

ROCK2 

Chk2/Rad53 

PKCβ 

skMLCK 

SSTK 

SNRK 

MAST4 MAST1 

DMPK 

NIM1 

CRIK 

DRAK1 TSSK3 

PKD2/PKCμ 

TSSK1 

SgK085 

TSSK2 

DMPK2 

caMLCK 

PKD1 

DCAMKL3 

DAPK2 

DAPK3 

DCAMKL1 

MELK PKD3/PKCν 

DAPK1 

DCAMKL2 

MRCKβ 

VACAMKL 

MRCKα 

MNK1 

PhKγ1 

MNK2 

PhKγ2 

PSKH1 

CaMKIIγ 

PSKH2 

BRSK2 

CaMKIIα 

CaMKIIβ 

BRSK1 

CaMKIIδ 

RSK4~b 

SNARK 

RSK1~b 

CaMKIV 

ARK5 

MSK2~b 

MSK1~b 

QSK 

RSK3~b 

RSK2~b 

CaMKIβ 

SIK 

QIK 

CaMKIγ 

MARK4 

CaMKIα 

CaMKIδ 

MARK3 

CHED 

DRAK2 

AMPKα2 

AMPKα1 

Haspin 

CASK 

MAPKAPK5 

MAPKAPK2 

MAPKAPK3 

STLK3 

STRAD/STLK5 

STLK6 

GRK7 

HPK1 

GCK 

NRK/ZC4 

Mst1 

Mst2 

TNIK/ZC2 

MYO3A 

MYO3B 

YSK1 

Mst3 

HGK/ZC1 

MINK/ZC3 



Tyrosine Kinases and 

Associated Phosphatases 

Tyrosine Kinase Signaling 

Whether functioning as cell surface receptors or as internal effectors of cell signaling, tyosine kinases 

control multiple aspects of cell and organism growth, differentiation, and function. Approximately 20 

receptor tyrosine kinase (RTK) families and at least 9 distinct groups of nonreceptor tyrosine kinases 

have been identified in humans. Regardless of localization, all tyrosine kinases regulate target protein 

function through transfer of phosphate from ATP to the hydroxyl group of a target protein tyrosine. 

EGF stimulation results in 

phosphorylation of EGFR at Tyr1068. 

Phospho-EGF Receptor (Tyr1068) (D7A5) XP ® Rabbit mAb #3777: 

WB analysis of extracts of BxPC-3 cells, untreated or EGF-stimulated, 

using #3777 (upper) and EGF Receptor Antibody #2232 (lower). 

kDa 

200 

140 

100 

80 

200 

140 

100 

80 

+ 

– 

Phospho- 

EGFR 

(Tyr1068) 

EGFR 

EGF 

The tyrosine kinase family, a distinct 

group within the human kinome 

CMGC 

CLK4 

CLK2 CLK1 

TK 

CDK8 CDK11 

MARK1 

MARK2 

Receptor Tyrosine Kinases 

Most RTKs are single-pass, transmembrane 

proteins that bind extracellular polypeptide ligands 

(i.e. growth factors) and cytoplasmic effector and 

adaptor proteins to regulate biological processes. 

Ligand binding promotes receptor dimerization and 

autophosphorylation of receptor tyrosine residues. 

The resultant conformational change stabilizes the 

active kinase, and subsequent phosphorylation 

events form binding sites for downstream adaptor, 

scaffold, and effector proteins. 

EGFR phosphorylation at multiple 

tyrosine residues provides binding 

sites for adaptor proteins and 

downstream signaling nodes. 

Trad Trio 

CAMK 

FGFR2 FGFR3 

TrkC 

EphB2 

TrkB 

FGFR1 

FGFR4 

EphB1 

TrkA 

FLT1/VEGFR1 

EphA5 

KDR/VEGFR2 

MuSK ROR2 

Fms/CSFR 

ROR1 

EphB3 

Ret 

Kit 

EphA3 

DDR2 

Mer 

EphA4 

DDR1 

Tyro3/ 

Axl 

FLT4 

Sky 

PDGFRα 

EphA6 

IGF1R IRR 

FLT3 

PDGFRβ 

InsR 

Yes 

EphB4 

Met 

EGFR HER2/ErbB2 

Src 

Ron 

MLK3 

Ros 

EphA7 

ALK 

MLK1 

Lyn 

LTK 

Tie2 

Tie1 

HCK 

Fyn 

RYK 

HER4 

EphA8 

MLK4 

CCK4/PTK7 

MLK2 

Lck 

Fgr 

Ack Tnk1 Tyk2 

Jak1 

HER3 

Jak2 

EphA2 

Jak3 

BLK 

TKL 

ANKRD3 SgK288 

DLK 

Syk Zap70/SRK 

EphA1 

PYK2/FAK2 

LZK 

TKL 

FAK 

Lmr1 

ALK4 

ITK 

Lmr2 

C-Raf/Raf1 

FRK 

TGFβR1 

ZAK 

BRaf 

TEC 

EphB6 

KSR 

Srm 

RIPK2 

KSR2 

TXK 

Brk 

BTK 

Lmr3 

ALK7 

IRAK3 

IRAK1 

LIMK1 

ARaf 

BMPR1B 

Etk/BMX 

EphA10 


BMPR1A 

CTK 

RIPK3 

TSK2 

TAK1 

HH498 

ALK1 

CSK 

ALK2 

Abl2/Arg 

IRAK2 

ActR2 

Abl Fes 

ActR2B 

Fer 

RIPK1 

Jak3~b 

TGFβR2 

LRRK2 

MEKK2/MAP3K2 

Jak2~b 

LRRK1 

MISR2 

MEKK3/MAP3K3 

Tyk2~b 

SuRTK106 

IRAK4 

BMPR2 

ASK/MAP3K5 

ANPα/NPR1 

MAP3K8 

Jak1~b 

MAP3K7 

ANPβ/NPR2 

NRBP1 NRBP2 MEKK1/MAP3K1 

KHS1 

MOS 

KHS2 

HSER 

SgK496 

WNK1 

STE 

WNK3 

MEKK6/MAP3K6 

DYRK2 

Mst4 

PBK 

MAP3K4 

DYRK3 

GUCY2D 

WNK2 

DYRK4 

DYRK1A 

NRBP1 

GUCY2F 

NRBP2 MEKK1/MAP3K1 OSR1 

DYRK1B 

WNK4 

MLKL 

PERK/PEK 

SgK307 

SLK 

PKR 

LOK 

HIPK1 HIPK3 

GCN2 SgK424 

TAO1 

SCYL3 TAO2 

HIPK2 

SCYL1 

Tpl2/COT 

SCYL2 

NIK 

TAO3 

PAK1 

CLK4 HIPK4 

PRP4 

HRI 

PAK3 

CLIK1 

PAK2 

IRE1 

CLK2 CLK1 

PAK4 

MAP2K5 

PAK5/PAK7 

CLIK1L 

IRE2 

CLK3 

TBCK 

PAK6 

MAP2K7 

MEK1/MAP2K1 

RNAseL 

GCN2~b 

MEK2/MAP2K2 

CK1 

TTK 

MSSK1 

SgK071 

SRPK2 

KIS 

MYT1 


SRPK1 

CK2α1 

Wee1 

MAK 

CK2α2 

SgK196 

CDC7 

Wee1B 

CK1δ 

ICK 

PRPK 

TTBK1 

CK1ε 

GSK3β 

AGC 

MOK 

TTBK2 

GSK3α 

CK1α1 

CDKL3 

CK1α2 

CDKL2 

PINK1 

SgK493 

SgK269 

VRK3 

CK1γ2 

CDKL1 CDKL5 

ERK7 

SgK396 

SgK223 

CDKL4 Erk4 

Slob 

CK1γ1 

Erk3 

SgK110 

PIK3R4 

CK1γ3 

NLK 

SgK069 

Bub1 

Erk5 

SBK 

BubR1 

Erk1/ 

IKKα 

p44MAPK 

IKKβ 

VRK1 

CDK7 

IKKε 

VRK2 

Erk2/ 

PLK4 CK1 

PITSLRE 

TBK1/NAK 

p42MAPK p38γ 

MPSK1 

JNK1 

p38δ 

JNK2 

TLK2 

JNK3 CDK10 

GAK 

TLK1 

PLK3 

p38β 

AAK1 

CDK8 CDK11 

CAMKK1 

ULK3 

PLK1 

p38α 

PLK2 

CDK4 

CCRK 

BIKE 

CAMKK2 

BARK1/GRK2 

CDK6 

CMGC 

ULK1 


Fused 

GRK5 

PFTAIRE2 

SgK494 

ULK2 ULK4 

GRK6 GRK4 

PFTAIRE1 

CDK9 

Nek6 

RSKL1 

PCTAIRE2 

Nek7 Nek10 

SgK495 

PASK 

PDK1 

RSKL2 

Nek8 

MSK1 

RSK1/p90RSK 

PCTAIRE1 

CDK5 CRK7 

PCTAIRE3 

Nek9 

LKB1 

MSK2 

RSK4 RSK2 

Chk1 

p70S6K 

RSK3 

Nek2 

Akt2/PKBβ 

AurA/Aur2 

p70S6Kβ 

Akt1/PKBα 

cdc2/CDK1 

Nek11 

Akt3/PKBγ 

CDK3 

Nek4 

AurB/Aur1 

SGK1 

CDK2 

AurC/Aur3 

SGK2 

SGK3 

Trb3 Pim1 Pim2 

PKG2 

PKN1/PRK1 

LATS1 

Nek3 

PKG1 

PKN2/PRK2 

Trb2 

Pim3 

PKN3 

Nek5 

Trad Trio 

LATS2 

NDR1 

PRKY 

PKCδ 

Trb1 

Obscn~b 

NDR2 

PKCθ 

PRKX 

YANK1 

SPEG~b 

MAST3 

PKCη 

Nek1 

MASTL 

PKCε 

Obscn 

STK33 

YANK2 

PKCι 

YANK3 

PKCζ 

SPEG 

PKAγ 

PKAα 

PKCγ 

TTN 

MAST2 

PKAβ 

smMLCK 

TSSK4 

ROCK1 

PKCα 

HUNK 

ROCK2 

Chk2/Rad53 

PKCβ 

skMLCK 

SSTK 

SNRK 

MAST4 MAST1 

DMPK 

NIM1 

CRIK 

DRAK1 TSSK3 

PKD2/PKCμ 

TSSK1 

SgK085 

TSSK2 

DMPK2 

EGF 

caMLCK 

PKD1 

DCAMKL3 

DAPK2 

DAPK3 

DCAMKL1 

MELK PKD3/PKCν 

DAPK1 

DCAMKL2 

MRCKβ 

VACAMKL 

MRCKα 

MNK1 

PhKγ1 

MNK2 

PhKγ2 

PSKH1 

CaMKIIγ 

PSKH2 

BRSK2 

CaMKIIα 

CaMKIIβ 

NH 2 

BRSK1 

CaMKIIδ 

RSK4~b 

SNARK 

RSK1~b 

CaMKIV 

ARK5 

MSK2~b 

MSK1~b 

CaMKII 

CaMKII 

Cys 

rich 

CHED 

Tyr845 

Tyr974 

Tyr992 

Tyr1045 

Ser1046 

Ser1047 

Ser1057 

Tyr1068 

Tyr1086 

Tyr1101 

Ser1142 

Tyr1148 

Tyr1173 

TK 

Src phosphorylation 

Autophosphorylation 

kinase domain 

kinase domain 

COOH 

EGFR homodimer 

P 

P 

P 

P 

P 

P 

P 

P 

P 

P 

P 

P 

P 

STAT5b 

AP-2 

PLCγ 

Cbl 

ubiquitination 

Grb2 

Gab1 


SHC 

SHP1 

PLCγ 

DRAK2 

CAMK 

DNA synthesis 

Translocation to 

mitochondria survival 

PKC 


p85 

PKC 

AMPKα2 

AMPKα1 

QSK 

SIK 

QIK 

degradation 

degradation 

MARK4 

MARK3 

MARK1 

MARK2 

MAPK/ERK 

Cascade 

degradation 

MAPK/ERK 

Cascade 

AKT/PKB 

Cascade 

MAPK/ERK 

Cascade 

Haspin 

RSK3~b 

RSK2~b 

CASK 

MAPKAPK5 

MAPKAPK2 

MAPKAPK3 

STLK3 

STRAD/STLK5 

STLK6 

CaMKIβ 

CaMKIγ 

CaMKIα 

CaMKIδ 

GRK7 

HPK1 

GCK 

NRK/ZC4 

Mst1 

Mst2 

TNIK/ZC2 

MYO3A 

MYO3B 

YSK1 

Mst3 

RTKs regulate a broad range of biological effects including cell growth, proliferation, survival, migration, 

STE and differentiation. For this reason, altered levels and/or activities of RTKs are commonly associated 

with many forms of human cancer. RTK activity can be chemically manipulated through the use of 

targeted, small-molecule inhibitors. 

HGK/ZC1 

MINK/ZC3 

Phospho-EGF Receptor (Tyr1068) (D7A5) 

XP ® Rabbit mAb #3777: Confocal IF analysis 

of HeLa cells, untreated (left) or EGF-treated 

(right), using #3777 (green). Blue pseudocolor 


HER2, an ErbB/HER family member, is 

overexpressed in 40% of human breast cancers. 

AGC 

HER2/ErbB2 (D8F12) XP ® Rabbit mAb #4290: IHC analysis of paraffin-embedded 

human breast carcinoma using #4290. 

ROS1, an RTK similar in structure to ALK that 

forms oncogenic fusion proteins with FIG1, 

SLC34A2, and CD74 

ROS1 (D4D6 ® ) Rabbit mAb #3287: IHC analysis of paraffin-embedded human lung 

carcinoma using #3287. Note: Staining is of FIG-ROS1 fusion. 

HER3/ErbB3 is overexpressed in many forms 

of cancer. 

HER3/ErbB3 (D22C5) XP ® Rabbit mAb #12708: IHC analysis of paraffin-embedded 

non-small cell lung carcinoma using #12708. 

Ligand activation 

of EGFR at the cell 

membrane results 

in phosphorylation 

at Tyr1068. 

Tyrosine Kinase 

Inhibitors 

#4401 Crizotinib 

Inhibitor of ALK and ROS1 

(ATP-competitive inhibitor) 

#9052 Dasatinib 

Tyrosine kinase inhibitor 

(Abl, BCR/Abl, Src, c-KIT, 

Ephrin receptors) 

#5083 Erlotinib 

Inhibits EGFR 


#4765 Gefitinib 

Inhibits EGFR 

(active site inhibitor) 

#9084 Imatinib 

Inhibits Bcr-Abl, PDGFR, 

c-Kit (active site inhibitor) 

#12121 Lapatinib 

Inhibits EGFR and HER2 


#12209 Nilotinib 

Tyrosine kinase inhibitor 

(Abl, BCR/Abl, c-KIT, LCK, 

Ephrin receptors, DDR1/2, 

PDGFR-B) 

#9493 PKC412 

Very broad spectrum protein 

kinase inhibitor (conventional 

PKCs, some RTKs, Syk, 

Cdk1/B, c-Src) 

#8705 Sorafenib 

Inhibits VEGFR and PDGFR; 

inhibits Raf kinases; Induces 

autophagy 

#12328 Sunitinib 

Broad RTK inhibitor 

(PDGFR, VEGFR, c-KIT, RET, 

CSF-1R, FLT-3/CD135) 

#9842 Tyrphostin AG 1478 

Inhibits EGFR 

#12998 Vatalanib 

Broad RTK inhibitor 

(VEGFR, PDGFR-B, c-KIT) 


www.cellsignal.com/csttyrosine 

67



ALK, an RTK that forms oncogenic fusion proteins with 

EML4, TFG, and KIF5B, is expressed in lung carcinoma. 

ALK (D5F3 ® ) XP ® Rabbit mAb #3633: IHC 

analysis of paraffin-embedded human lung 

carcinoma with high (left) and low (right) levels 

of ALK expression using #3633. 

Phospho-Jak2 (Tyr1008) (D4A8) Rabbit 

mAb #8082: WB analysis of extracts from 

BaF3 cells (A), untreated or treated with Mouse 

Interleukin-3 (mIL-3) #8923 (10 ng/ml, 5 min), 

using #8082 (upper) or total Jak2 (D2E12) XP ® 


Jak2 (D2E12) XP ® Rabbit mAb #3230: 

Confocal IF analysis of K-562 cells (B) using 

#3230 (green). Blue pseudocolor = DRAQ5 ® 


A 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

200 

140 

100 

Phospho- 

Jak2 

(Tyr1008) 

Jak2 

B 

Treatment with 

IL-3 results in 

phosphorylation 

of Jak2 at Tyr1008. 

80 

60 

50 

40 

Ligand activation of 

Met, a high affinity RTK 

for hepatocyte growth 

factor (HGF), results 

in phosphorylation at 

Tyr1234/1235. 

A kDa 

200 

140 

100 

80 

60 

50 

40 

Phospho-Met 

(Tyr1234/5) 

B 

Phospho-Met (Tyr1234/1235) (D26) XP ® Rabbit mAb #3077: WB analysis of cell 

extracts from HeLa cells, untreated or stimulated with HGF (A), using #3077 (upper) and Met 

(25H2) Mouse mAb #3127 (lower). Confocal IF analysis of MKN-45 cells, untreated (B) or 

treated with SU11274 (1 μM, 3 hr) (C), using #3077. Blue pseudocolor = DRAQ5 ® #4084 


C 

Treatment with a 

Met inhibitor prevents 

Met activation and 

phosphorylation at 

Tyr1234/1235. 

The nonreceptor tyrosine kinase 

Zap-70 is expressed in T cells. 

30 

– + 

Zap-70 (D1C10E) XP ® Rabbit mAb (Alexa Fluor ® 488 Conjugate) #9473: 

Flow cytometric analysis of Ramos (B cells; blue) and Jurkat (T cells; green) cells 

using #9473. 

mIL-3 

Events 

Zap-70 

200 

140 

100 

80 

60 

50 

40 

– + 

Met 

HGF 

Nonreceptor Tyrosine Kinases 

The cytoplasmic nonreceptor tyrosine kinases transduce signals generated by RTKs and other receptors 

from the cell surface to the nucleus. Receptor-associated tyrosine kinases are phosphorylated within the 

activation loop of their kinase catalytic domain, resulting in activation and propagation of downstream 

signals through phosphorylation of other kinases, adaptor proteins, or transcription factors. In addition 

to their kinase domain, nonreceptor tyrosine kinases often contain various domains that mediate 

protein-protein or protein-lipid interactions, which allows association with activated receptors or downstream 

signaling nodes. For example, SH2 domains bind phospho-tyrosine residues, while PH domains 

direct interactions with membrane lipids. 

PDGF treatment results in phosphorylation 

of Src family proteins at Tyr416. 

Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb #6943: WB analysis of extracts 

NIH/3T3 cells, serum-starved or treated with human Platelet-Derived Growth Factor BB 

hPDGF-BB #8912 (100 ng/ml, 15 min), using #6943 (upper) or Src (36D10) Rabbit mAb 

#2109 (lower). 


Arteaga, C.L. and Engelman, J.A. (2014) Cancer Cell 25, 282−303. • Davies, K.D. and Doebele, R.C. (2013) Clin. Cancer 

Res. 19, 4040−4045. • Hallberg, B. and Palmer, R.H. (2013) Nat. Rev. Cancer 13, 685−700. • Liu, S.T., Pham, H., Pandol, 

S.J., and Ptasznik, A. (2014) Front. Physiol. 4, 416. • Miaczynska, M. (2013) Cold Spring Harb. Perspect. Biol. 5, a009035. 

• Panjarian, S., Lacob, R.E., Chen, S., Engen, J.R., and Smithgall, T.E. (2013) J. Biol. Chem. 288, 5443−5450. • Shaw, A.T., 

Hsu, P.P., Awad, M.M., and Engelman, J.A. (2013) Nat. Rev. Cancer 13, 772−787. 

kDa 

100 

80 

60 

50 

40 

30 

20 

100 

80 

60 

50 

40 

30 

20 

– + 

Phospho-Src 

(Tyr416) 

Src 

hPDGF-BB 

Commonly Studied Tyrosine Kinases Targets 


ACPP 

• 

ALK • • • 

Phospho-ALK (Tyr1078) • • 



Phospho-ALK (Tyr1282/Tyr1283) • • 

• 

Phospho-ALK (Tyr1278/Tyr1282/ 

Tyr1283) 

Phospho-ALK (Tyr1586) • • • 

Phospho-ALK (Tyr1604) • • • 

Axl 

• • • • 

Phospho-Axl (pan Tyr) 

• 

Phospho-Axl (Tyr702) • 

DDR1 

• • • 

Phospho-DDR1 (pan Tyr) 

• 

Phospho-DDR1 (Tyr792) • 

DDR2 

• 

EGFR 

• • • • • 

Phospho-EGFR (Thr669) • • 

Phospho-EGFR (Tyr845) • • • 

Phospho-EGFR (Tyr992) • 



Phospho-EGFR (Ser1046/1047) • 

Phospho-EGFR (Tyr1068) • • • • 



Phospho-EGFR (Tyr1173) • • 

• • • 

EGF Receptor 

(E746-A750del Specific) 


EGF Receptor (L858R Mutant Specific) • • 

EphA2 

• 

Phospho-EphA2 (Tyr588) • 



Phospho-EphA2 (Ser897) • 


EphA3/EphA4/EphA5 • 

EphB1 

• 

EphB6 

• 

Phospho-Ephrin B (Tyr324/329) • 

Eps8 

• 

EREG 

• 

Etk/BMX 

• 

Phospho-Etk/BMX (Tyr40) • 

FGFR1 • • • • 

FGFR1 (pan Tyr) 

• 

FGFR1 (Tyr653/Tyr654) • • • 

FGFR1 (Tyr766) 

• 

FGFR2 • • • 

Phospho-FGFR2 (Pan Tyr) 

• 

FGFR3 

• 

FGFR4 • • • • 

FLT3 • • 

Phospho-FLT3 (pan Tyr) 

• 

Phospho-FLT3 (Tyr589/Tyr591) • 

Phospho-FLT3 (Tyr591) • • • 

Phospho-FLT3 (Tyr842) • 

Phospho-FLT3 (Tyr969) • 

HER2/ErbB2 

• • • • • 


nodes within tyrosine kinases signaling 


in tyrosine kinases research. Primary 












www.cellsignal.com/csttyrosine 69



181 


CST antibodies for EGFR have been 

cited over 181 times in high-impact, 

peer-reviewed publications from the 

global research community. 


Phospho-HER2/ErbB2 (pan Tyr) • 

Phospho-HER2/ErbB2 (Tyr877) • 


• • • 

Phospho-HER2/ErbB2 

(Tyr1221/1222) 

Phospho-HER2/ErbB2 (Tyr1248) 

Phospho-HER2/ErbB2 

(Tyr1248)/EGFR (Tyr1173) 

• 

• 

HER3/ErbB3 • • • • 

Phospho-HER3/ErbB3 (pan Tyr) • 





HER4/ErbB4 • • 

Phospho-HER4/ErbB4 (Pan Tyr) • 



c-Kit • • • • 

Phospho-c-Kit (pan Tyr) 

• 

Phospho-c-Kit (Tyr703) • 

Phospho-c-Kit (Tyr719) • • 

LRIG1 

• 

M-CSF Receptor 

• • 

Phospho-M-CSF Receptor (Tyr546) • 

Phospho-M-CSF Receptor (Tyr699) • • 


Phospho-M-CSF Receptor (Tyr723) • • 



Mer • • 

Met 

• • • • • 

Phospho-Met (pan Tyr) 

• 

Phospho-Met (Tyr1003) • • 

Phospho-Met (Tyr1234/1235) • • • • 

Phospho-Met (Tyr1349) • • • 

PDGFR-α • • • • 


Wu, J. et al. (2014) EGFR-STAT3 signaling promotes formation 

of malignant peripheral nerve sheath tumors. Oncogene 

33, 173–180. 

Sheu, J.J. et al. (2014) LRIG1 modulates aggressiveness of 

head and neck cancers by regulating EGFR-MAPK-SPHK1 

signaling and extracellular matrix remodeling. Oncogene 33, 

1375–1384. 

Bronisz, A. et al. (2014) xtracellular vesicles modulate the 

glioblastoma microenvironment via a tumor suppression 

signaling network directed by miR-1. Cancer Res. 74, 

738–750. 

De Cesare, M. et al. (2014) Synergistic antitumor activity 

of cetuximab and namitecan in human squamous cell 

carcinoma models relies on cooperative inhibition of EGFR 

expression and depends on high EGFR gene copy number. 

Clin. Cancer Res. 20, 995–1006. 

Muller, P.A. et al. (2014) Mutant p53 regulates Dicer through 

p63-dependent and -independent mechanisms to promote 

an invasive phenotype. J. Biol. Chem. 289, 122–132. 


Phospho-PDGFR-α (Tyr754) • 




Phospho-PDGF Receptor α/β (pan Tyr) • 

Phospho-PDGF Receptor α • 

(Tyr849)/PDGF Receptor β (Tyr857) 

PDGFR-β • • • 

Phospho-PDGFR-β (Tyr740) • 

Phospho-PDGFR-β (Tyr751) • • • 




PTK7 

• 

Ret 

• • • 

Phospho-Ret (pan Tyr) 

• 

Phospho-Ret (Tyr905) 

• 

Ron • • 

ROR1 

• 

ROR2 

• 

ROS1 • • • 

Phospho-ROS1 (pan Tyr) 

• 

Phospho-ROS1 (Tyr2274) • 

Spry1 

• 

Tie2 

• 

Phospho-Tie2 (Tyr992) • 

Phospho-Tie2 (Ser1119) • 

Tyro3 

• 

VEGFR1 

• 

VEGFR2 • • • • 

Phospho-VEGFR2 (Tyr951) • • 

Phospho-VEGFR2 (Tyr996) • 


Phospho-VEGFR2 (Tyr1175) • • 


VEGFR3 

• • 

Stahlschmidt, W. et al. (2014) Clathrin terminal domainligand 

interactions regulate sorting of mannose 6-phosphate 

receptors mediated by AP-1 and GGA adaptors. J Biol Chem. 

21, 4906–4918. 

Tao, J.J. et al. (2014) Antagonism of EGFR and HER3 

enhances the response to inhibitors of the PI3K-Akt pathway 

in triple-negative breast cancer. Sci. Signal. 7, ra29. 

Zhang, F. et al. (2014) Temporal production of the signaling 

lipid phosphatidic acid by phospholipase D2 determines the 

output of extracellular signal-regulated kinase signaling in 

cancer cells. Mol. Cell Biol. 34, 84–95. 

Lee, C.Y. et al. (2014) Neuregulin autocrine signaling 

promotes self-renewal of breast tumor-initiating cells by triggering 

HER2/HER3 activation. Cancer Res. 74, 341–352. 

Bronisz, A. et al. (2014) Extracellular vesicles modulate the 

glioblastoma microenvironment via a tumor suppression 

signaling network directed by miR-1. Cancer Res. 74, 

738–750. 

Tyrosine Kinases Kinase-Disease Associations 

Name Group Disease Type Molecular Notes 

ALK TK Cancer Trans About one third of large-cell lymphomas are caused by a t(2;5)(p23;q35) translocation 

that fuses ALK to nucleophosmin (NPM1A). Other cases caused by fusions of ALK to 

moesin, non-muscle myosin heavy chain 9, clathrin heavy chain and other genes. 

Several fusions also seen in inflammatory myofibroblastic tumors and expression 

has been briefly noted in a range of tumors (Medline:15095281). Proposed as tumor 

antigen (Medline:11877285). OMIM:105590. 

ALK1 

(ACVRL1) 

ALK2 

(ACVR1) 

ALK4 

(ACVR1B) 

TKL Cardiovascular Mut Fourteen distinct mutations linked to hereditary hemorrhagic telangiectasia type 2 

(Osler-Rendu-Weber syndrome 2) [OMIM:600376], associated with intestinal bleeding, 

arterial hypertension and arteriovenous malformations. OMIM:601284. 

TKL Development Mut Single heterozygous mutation seen in many independent cases of fibrodysplasia ossificans 

progressiva [OMIM:135100], causing skeletal malformations and extra-skeletal 

bone formation. OMIM:102576. 

TKL Cancer Mut, Splice Two somatic truncation mutations seen in pancreatic carcinoma. Unique splice forms 

with predicted dominant negative activity. OMIM:601300. 

Axl TK Cancer OE Overexpression in tissue culture causes oncogenic transformation. Overexpressed in 

several cancers including thyroid (Medline:10411118), ovarian (Medline:15452374), 

gastric (Medline:12168903), ER+ breast cancer (Medline:11484958) and acute 

myeloid leukemia, where it is associated with poor prognosis (Medline:10482985). 

OMIM:109135. 

BTK TK Cancer, 

Immunity 

LOF Mut 

EGFR TK Cancer Amp, OE, 

GOF Mut 

Eph 

family 

TK 

Cancer, 

Sensory 

LOF mutations cause X-linked agammaglobulinemia [OMIM:300300], arresting 

development of B cells and causing recurrent bacterial infections. Truncated splice 

forms found in childhood leukemias may underlie radiation resistance of tumors 

through inhibition of apoptosis (Medline: 12854903). Inhibitors developed to target B 

cell maturation and function. Inhibitor: dasatinib. OMIM:300300. 

Overexpressed in breast, head and neck cancers (Medline:15254682) and correlated 

with poor survival. Activating somatic mutations seen in lung cancer, corresponding 

to minority of patients with strong response to EGFR inhibitor gefinitib. Mutations and 

amplification also seen in glioblastoma, and upregulation seen in colon cancer and 

neoplasms. In xenografts, inhibitors synergized with cytotoxic drugs in inhibition of many 

tumor types (Medline:10815932). Inhibitors: gefinitib/ZD1839 (Astra Zeneca), cetuximab 

(mAb, Imclone), erlotinib (OSI/Genentech) lapatinib (Glaxo Smith Kline). OMIM:131550. 

A 14-member family of receptor tyrosine kinases with similar functions in intercellular 

communication, migration, patterning and angiogenesis. Ephrins are implicated in 

development of tumor vasculature and intercellular contacts required for metastasis. 

Several members are overexpressed in cancers. Soluble forms (competitive receptors) 

have shown some anti-tumor activity and extracellular domains have been used as 

tumor-specific antigens. 

EphA1 TK Cancer Expr Misexpressed in several cancers and upregulated in head and neck cancer 

(Medline:15023838). Downregulated in invasive breast cancer cell lines (Medline:15147954) 

and glioblastoma (Medline:14726470). OMIM:179610. 

EphA2 TK Cancer OE Overexpressed in many cancers including aggressive ovarian (Medline:15297418), 

cervical (Medline:15297167), breast carcinomas (Medline: 15147954) and lung 

cancer (Medline:12576426). Expression correlates with degree of angiogenesis 

(Medline:14965363), metastasis (Medline: 14767510) and xenograft tumor growth 

(Medline:14973554). Soluble receptor inhibits tumor growth and angiogenesis in mice 

(Medline:12370823, 14670182). OMIM:176946. 

EphA3 

(HEK) 

TK Cancer Mut Two point mutations seen in a survey of colorectal tumors (Medline:12738854). 

Soluble receptors reduce tumor growth and angiogenesis in mouse models (Medline:12370823, 

14670182). Nine point mutations found in 294 colon and lung tumors 

(Medline:16941478) OMIM:179611. 

EphB2 TK Cancer OE, Mut Point mutations seen in prostate cancer (Medline:15300251). Overexpressed and 

required for migration of glioblastoma (Medline:15126357). Overexpressed and correlated 

with poor survival in breast cancer (Medline:15029258). Overexpression and loss 

of heterozygosity seen in colorectal cancers (Medline:11920461, 11166921). Target 

for immunoconjugate drug therapy (Medline:14871799). OMIM:600997. 

EphB4 

(HTK) 

TK Cancer OE Required for normal development and angiogenesis of the mammary gland. Angiogenic 

functions may be kinase-independent by means of retrograde signaling through its 

ephrin-B2 ligand (Medline:15067119). High expression correlates with malignancy in 

breast, ovarian and other cancers, but appears to be a survival factor (Medline:16816380, 

17353927). Also upregulated in head and neck (Medline:14661437), 

endometrial (Medline:12562648) and colon carcinomas (Medline:11801186). 

OMIM:600011. 

FGFR1 TK Cancer, 

Development 

Mut, Trans 

Point mutations cause Pfeffer syndrome [OMIM:101600] (finger and toe malformations 

and other skeletal errors) and dominant Kallmann syndrome 2 [OMIM:147950]. Stem 

cell leukemia lymphoma syndrome (SCLL) may be caused by a t(8;13)(p12;q12) 

translocation that fuses a zinc finger gene (ZNF198) to FGFR1. Various myeloproliferative 

disorders have been linked to translocations that fuse FGFR1 to FOP, FIM, CEP1 or 

the atypical kinase, BCR. Inhibitor: SU5402. OMIM:136350. 







Development 

Mut, Amp Mutations cause syndromes with defects in facial and limb development, including 

Crouzon syndrome [OMIM:123500], Beare-Stevenson cutis gyrata syndrome 

[OMIM:123790], Pfeiffer syndrome [OMIM:101600], Apert syndrome [OMIM:101200], 

and Jackson-Weiss syndrome [OMIM:123150]. Somatic mutations seen in gastric 

cancer (Medline:11325814). Amplified in gastric (Medline:14595756), breast 

(Medline:11564899) and some B cell cancers (Medline:12203778), but deleted in 

glioblastoma (Medline:14756442). Promoter SNP associated with breast cancer occurrance 

(Medline: 17529967). OMIM:176943. 


Development 

GOF Mut, 

Trans 

Activating point mutations cause dwarfism, including achondroplasia [OMIM:100800], 

hypochrondroplasia [OMIM:146000] and thanatophoric dysplasia [OMIM:187600]; facial 

and other morphogenetic disorders, including Crouzon syndrome [OMIM:612247], 

craniosynostosis Adelaide type [OMIM:600593], San Diego skeletal displasia 

[OMIM:187600] and Muenke syndrome [OMIM:602849]. Translocations t(4;14) involving 

the IgH region are common in multiple myeloma and frequently involve FGFR3. 

Activated FGFR3 found in 30% of bladder cancers and several cervical cancers but not 

in other tumors. Two mutations found in colorectal cancer. OMIM:134934. 

FGFR4 TK Cancer SNP A common SNP variant associated with increased motility and progression of breast 

cancer (Medline:11830541, but see also Medline:14710228), head and neck cancer 

(Medline:15197773) and soft tissue sarcomas (Medline:14601095). Increased 

expression seen in pituitary adenomas, pancreatic cancer and breast cancer cell lines. 

OMIM:134935. 

FLT1 

(VEGFR1) 

TK Cancer Meth, OE Angiogenesis modulator that may both co-operate with and antagonize KDR/VEGFR2 

(Medline:14984769). Overexpressed in several tumor types (Medline:12681367, 

9582527, 10738243, 10893635) and an antagonistic soluble form is inhibited in 

progressive tumors (Medline:15112269, 14605010, 15173272). Downregulated 

by hypermethylation in prostate cancer (Medline:12824880). The soluble receptor 

and mutant forms have anti-tumor activity in model systems (Medline:15221961, 

15126877). Inhibitors: SU11248, PKC412, CEP-5214. OMIM:165070. 

FLT3 TK Cancer GOF Mut Activating mutations found in one third of cases of acute myeloid leukemia (AML), as 

well as in acute lymphoblastic leukemia, acute promyelocytic leukemia and myelodysplastic 

syndrome. Inhibitors: SU11248 and PKC412. OMIM:136351. 

FLT4 

(VEGFR3) 

TK 

Lymphangiogenesis 

Fyn TK Cancer, 

Epilepsy 

HER2 

(ErbB2) 

Act, LOF 

Mut 

Lymphatic-specific VEGF receptor. LOF mutations cause hereditary lymphedema 

[OMIM:153100], and one case of capillary infantile hemangioma [OMIM:602089]. 

Expression of FLT4 ligands is seen in a variety of tumors, including colorectal, prostate, 

gastric, breast and thyroid cancers where it is associated with lymph node metastasis 

(Medline:19787226, 14614015, 14534690, 10430087, 14716745, 15107801). In 

model systems, overexpression of ligand increases lymph node metastasis and blocking 

antibodies and soluble receptors have been used to reduce lymphangiogenesis 

(Medline:15072591, 11175849). Inhibitors: BAY 43-9006, CEP-7055. OMIM:136352. 

Induced expression aids in cellular transformation and xenograft metastasis (Medline:3287380, 

8325712). In squamous cell carcinoma, Fyn transduces signals from 

EGFR and Src and is required for cell migration and invasiveness (Medline:11684709). 

Activity linked to migration in a murine melanoma model (Medline:13129922). Appears 

to block late stage development of neuroblastoma (Medline:12450793). Mouse knockout 

deficient in kindling response, a model for human epilepsy. OMIM:137025. 

TK Cancer Amp, OE EGF family receptor. Overexpression induces constitutive activity and the gene is amplified 

or overexpressed in up to 30% of breast cancers, correlating with poor survival. 

The antibody trastuzumab is approved for treatment of metastatic breast cancer with 

HER2 amplification/overexpression. Somatic mutations seen in 4% of lung cancers and 

also in breast, gastric, ovarian cancer and glioblastoma. One SNP shows predisposition 

to breast and gastric cancer (Medline:10699071, 14520697). Inhibitors: trastuzumab 

(mAb, Genentech), lapatinib (Glaxo Smith Kline), PKI-166 (Novartis), EKB-569, CI-1033. 

OMIM:164870. 


Met TK Cancer GOF Mut, 

OE, Trans 

PDGFRα TK Cancer, 

Development 

Trans, Del, 

Mut 

Activating point mutations cause hereditary papillary renal carcinoma [OMIM:605074]. 

Mutations also seen in sporadic renal cell carcinoma and childhood hepatocellular 

carcinoma. Upregulation in carcinomas and sarcomas correlates with metastasis and 

poor outcome (Medline:14617781). Some gastric carcinomas harbor a translocation 

that creates an activated TPR-Met fusion protein (Medline:2052572). A small molecule 

inhibitor (PHA-665752) shows an effect in gastric carcinoma xenografts (Medline:14612533). 

Inhibitors: SU11274, PHA-665752, MGCD265 mAbs. OMIM:164860. 

Chromosomal rearrangments activate PDGFRa by fusion to Bcr causing atypical chronic 

myelogenous leukemia (CML), and to FIP1L1 causing idiopathic hypereosinophilic 

syndrome [OMIM:607685]. Activating point mutations cause a minority of gastrointestinal 

stromal tumors (GIST). Promoter polymorphisms linked to neural tube defects 

including spina bifida (Medline:11175793) as verified by mouse mutant model 

(Medline:9826722). Inhibitors: imatinib, SU11248. OMIM:173490. 

PDGFRβ TK Cancer Trans, OE A variety of myeloproliferative disorders and cancers result from translocations that activate 

PDGFRb by fusion with proteins such as TEL/ETV6, H2, CEV14/TRP11, rabaptin 

5 and huntington interacting protein 1. Imatinib treatment of TEL fusions has been 

successful. PDGFRb is also overexpressed in metastatic medulloblastoma. Inhibitors: 

imatinib, SU11248. OMIM:173410. 

Ret TK Cancer, 

Development 

GOF Mut, 

LOF Mut, 

Trans 

Familial GOF mutations cause endocrine cancers including familial medullary 

thyroid carcinoma (FMTC) [OMIM:155240], multiple neoplasia type IIA (MEN2A) 

[OMIM:171400] and MEN2B [OMIM:162300], both of which are characterized by predisposition 

to FMTC and phaeochromocytoma. Translocation-mediated fusion of Ret to 

various genes (H4, ELE1, PKA-R1, TIF1A, TIF1G) results in papillary thyroid carcinoma 

[OMIM:188550]. Familial LOF mutations cause Hirschsprung disease [OMIM:142623] 

in which enteric (intestinal) neurons fail to develop. OMIM:164761. 

RON TK Cancer OE, Splice Functions in cell migration and epithelial-mesenchymal transition. Highly expressed 

in tumors including head and neck cancer (Medline:15023838), colon (Medline:12527888), 

breast (Medline:9671413) ovarian carcinoma (Medline:12915129) 

and renal oncocytoma (Medline:15252311). Unusual splice variants seen in 

cancer, including a constitutively active form lacking the extracellular domain 

(Medline:12527888, 15289319). Overexpression in mouse lung leads to pulmonary 

adenomas (Medline:12214279). OMIM:600168. 

SRC TK Cancer Mut, OE, 

Act 

Tie2 TK Angiogenesis, 

Cancer 

Mut, OE 

Homolog of Rous sarcoma virus v-src. A truncated, activated form seen in approximately 

12% of colon cancers in one study and in one endometrial sarcoma but not seen 

in several other populations (Medline:9988270, 10804287, 10704743, 10485460, 

11161376). Expression and kinase activity are frequently increased in a wide array of 

cancers, including tumors from breast, colon, pancreas, lung, ovary and CNS (Medline:9988270). 

Inhibitors: SU6656 (Sugen), PD173955, PD166285 (Pfizer), CGP76030 

(Novartis), BMS-354825 (Bristol Myers Squibb; Phase 2 cancer). OMIM:190090. 

Point mutations cause dominantly inherited venous malformations [OMIM:600195]. 

Expression is increased in non-small cell lung cancer (Medline:10499626), myeloid 

leukemia (Medline:11755466) and hepatocellular carcinoma (Medline:11915032). 

Expression is prognostic of metastasis in breast cancer (Medline:12527939, 

15026804) and expression and activation correlate with malignancy in astrocytomas 

(Medline:14742253). Soluble receptor used to inhibit tumor growth in mice (Medline:14985859). 

OMIM:600221. 

Yes TK Cancer Amp, Act Ortholog of Yamaguchi sarcoma virus v-yes oncogene, which can transform fibroblasts 

in vitro (Medline:3303862). Amplified in one case of gastric cancer and in canine 

mammary tumors (Medline:3935622, 10081762). Kinase activity is increased in colon 

carcinoma cell lines and tumors (Medline:7690925, 7806032) and in melanoma 

cell lines and brain metastases (Medline:7690926, 9681823). Mouse knockouts 

have no strong phenotype due to compensation by Src and Fyn (Medline:7958873). 

OMIM:164880. 

HER3 

(ErbB3) 

HER4 

(ErbB4) 

TK Cancer OE EGF family receptor. Kinase domain lacks activity but heterodimerizes with other EGFRs 

to transduce growth signals. May be required for HER2 activity (Medline:12853564). 

Elevated expression in breast and other tumors is indicative of poor outcome (Medline:12866037, 

12896906, 14614020, 15150091, 7656248). A secreted form is 

expressed in metastatic prostate cancer (Medline:15141384). OMIM:190151. 

TK Cancer Expr Heterodimerizes and signals with other EGF receptors. May act as a tumor suppressor 

and is overexpressed in head and neck cancer (Medline: 15476268) but downregulated 

in renal cancer (Medline:15360049), papillary carcinoma (Medline:15279891), highgrade 

gliomas (Medline:15148612) and invasive breast cancer (Medline:15084248). 

OMIM:600543. 

Zap-70 TK Immunity Mut Mutations cause selective T cell defect [OMIM:176947], a recessive form of severe 

combined immunodeficiency (SCID) exhibiting selective absence of CD8+ T cells. 

Reduced expression predicts positive outcome in B cell chronic lymphocytic leukemia. 

The SKG mouse is mutated at Zap-70, producing increased numbers of self-reactive T 

cells and resulting in chronic arthritis. OMIM:176947. 

Molecular: Act Activated • Amp Amplified • Del Deleted • Expr Expression • GOF Gain-of-function • Inh Inhibitor Studies • LOF Loss-offunction 

• LOH Loss-of-heterozygosity • Meth Methylation • Model model organism studies • Mut Mutation OE Overexpression • SNP Single 

Nucleotide Polymorphism • Splice Splicing change • Trans Translocation 

Kit TK Cancer, 

Depigmentation 

GOF Mut, 

LOF Mut, 

Act 

Activating mutations cause >90% of gastrointestinal stromal tumors (GIST) 

[OMIM:606764]; successfully treated with inhibitors imatinib and Sutent (Sutinib, 

SU11248). Activating mutations also induce mastocytosis [OMIM:154800] (Medline:15507672). 

Autocrine/paracrine stimulation may drive some lung and other tumors 

(Medline:15036937). Loss of expression associated with melanoma progression (Medline:9687504). 

Familial loss of function mutations cause piebaldism [OMIM:172800] 

with defects in hair and skin pigmentation due to lack of melanocytes. OMIM:164920. 

LYN TK Cancer Act Mouse knockout develops monocyte/macrophage tumors, while an activated transgene 

does not induce tumors. Hyperactivated in acute myeloid leukemia; treatment by 

antisense or drug inhibitors reduces proliferation (Medline:10360372). Lyn-specific 

inhibitors block proliferation in three prostate cancer cell lines (Medline:14871838). 

OMIM:165120. 

PhosphoSitePlus® PTM Research 

Please see pages 250–253 or visit our website for resources relating to PhosphoSitePlus ® PTM. 

www.cellsignal.com/exploration 



73

HER2/ICD 


ErbB/HER Signaling 

HER4/ErbB4 

PSD95 

Cytoplasm 

Nucleus 

Gab1 

Memo 

Cell 

Migration 

TACE 

γ-Sec 

BDP1 

Shc 

Cell 

Cycle 

Cell 

Adhesion 

HER2/ErbB2 

NRG4 NRG4 

CXCR4 

Invasion 

Metastasis 

Akt 

mTOR 

p70S6K 

HER4/ErbB4 

HER4/ErbB4 

HER2/ErbB2 

PI3K 

NRG2 NRG3 

Vav1 

Rac1 

NRG1 

HER3/ErbB3 

HER2/ErbB2 

Shc 

TNS3 

GRB2 

p130 Cas 

PAK1 

MEKK1 

JNK 

Importin β1 

Protein 

Nup358 

Synthesis 

Proliferation Apoptosis Survival 

Cellular Proliferation, 

DNA Repair 

cdc2 PCNA DNA-PK 

EGFR/ErbB1 

HER2/ErbB2 

Increase 

Cell Motility 

TNS4 

FAK 

Nck1 

Muc1 

cdc42 

β-Cat 

MIG6 

Cell 

Motility 

EGFR/ErbB1 

Caveolin 

EGF EGF 

Paxillin 

E-cadherin 

Cytoskeletal 

Regulation 

HB-EGF TGF-α 

Crk 

GRB2 

c-Abl 

Cbl 

PLC 

Calmodulin 

CaMK 

AREG 

Dok 

GAB2 

SHP-2 

AIP4 

Endosomemediated 

Recycling 

of EGFR 

PKC 

EREG 

GEP100 

Ub 

HRS 

MEK 

MAPK 

EPG 

Lysosomemediated 

Degradation 

of EGFR 

Calcineurin 

SOS 

Raf 

EGFR/ErbB1 

Ras 

βCEL. 

ARF6 

Yes 

Tumor 

Invasion 

Stat 

TSAd 

Src 

Syk 

Jak 

MEKK2/3 

MEK5 

ERK5 

MEF2 

G Protein-Coupled Receptors 

G protein-coupled receptors (GPCRs) constitute a large protein family that sense molecules outside 

the cell and activate signal transduction pathways inside the cell. Ligands that bind and activate these 

receptors include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, 

and vary in size from small molecules to peptides to large proteins. 

While the exact size of the GPCR superfamily is unknown, nearly 800 different human genes have been 

predicted to be a part of the GPCR superfamily from sequence analysis. About 350 of these detect hormones, 

growth factors, and other endogenous ligands. Approximately 150 of the GPCRs found in the 

human genome have unknown functions. Based on sequence homology and functional similarity, the 

GPCRs can be grouped into 6 classes. Class A, Rhodopsin-like receptors, comprise the largest class of 

nearly 85% of the genes. Over half of these are predicted to encode olfactory receptors while the rest 

are bound by endogenous compounds or are classified as orphan receptors. Class B genes comprise 

the secretin receptor family. Class C genes encode metabotropic receptors that bind neurotransmitters 

such as glutamate. Class D genes are defined by their fungal mating pheromone receptor functions. 

Class E genes are grouped based on cyclic AMP receptor-like function, and Class F genes are grouped 

based on Frizzled/Smoothened genotypes. 

Metabotropic glutamate receptor 1 (mGluR1) is a Class C GPCR 

in the mammalian brain for the neurotransmitter glutamate. 

A 

kDa 

200 

140 

1 2 

mGluR1 

B 

C 


Peptide ligand 

Neuropeptide Y is expressed 

in rat retina. 

Neuropeptide Y (D7Y5A) XP ® Rabbit 

mAb #11976: Confocal IF analysis of 

adult rat retina using #11976 (green). 



ICD 

HER4 

Stat 

ICD 

HER4 

TAB2 

N-CoR 

EGFR/ErbB-1 

EGFR/ErbB-1 

E2F Stat CREB Jun Fos Elk Stat 

MEF2 

100 

80 

Lanes 

1. neonatal mouse brain 

2. rat brain 

mGluR1 (D5H10) Rabbit mAb #12551: WB analysis of extracts from neonatal mouse 

brain and rat brain using #12551 (A). Confocal IF analysis of adult mouse cerebellum 

tissue 4X (B) and 20X (C) using #12551 (green). Blue pseudocolor = DRAQ5 ® #4084 


Cell Proliferation, Inflammation, 

Genome Instability, Tumor Progression 

Cell Growth/Differentiation, 

Cell Shape, Chemotaxis 

Growth Differentiation 

The ErbB receptor tyrosine kinase family consists of four cell surface receptors: ErbB1/ EGFR/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4. ErbB receptors are typical 

cell membrane receptor tyrosine kinases that are activated following ligand binding and receptor dimerization. Ligands can either display receptor specificity (i.e. EGF, TGF-α, 

AR, and Epigen bind EGFR) or bind to one or more related receptors; neuregulins 1–4 bind ErbB3 and ErbB4 while HB-EGF, epiregulin, and β-cellulin activate EGFR and 

ErbB4. ErbB2 lacks a known ligand, but recent structural studies suggest its structure resembles a ligand-activated state and favors dimerization. 

The ErbB receptors signal through Akt, MAPK, and many other pathways to regulate cell proliferation, migration, differentiation, apoptosis, and cell motility. ErbB family members 

and some of their ligands are often over-expressed, amplified, or mutated in many forms of cancer, making them important therapeutic targets. For example, researchers 

have found EGFR to be amplified and/or mutated in gliomas and NSCLC while ErbB2 amplifications are seen in breast, ovarian, bladder, NSCLC, as well as several other tumor 

types. Preclinical and clinical studies have shown that dual targeting of ErbB receptors display better efficacy than single treatment. 

Besides functioning as receptors on the cell surface, ErbB family proteins are also present in the nucleus to act as both kinases and transcriptional regulators. For example, 

EGFR could be transported into the nucleus where it functions as a tyrosine kinase to phosphorylate and stabilize PCNA. Similarly, membrane-bound ErbB2 interacts with 

importin β1 and Nup358 and migrates to the nucleus via endocytic vesicles. Inside the nucleus, ErbB2 modulates the transcription of multiple downstream genes including 

COX-2. In addition, NRG or TPA stimulation promotes ErbB4 cleavage by γ-secretase, releasing an 80 kDa intracellular domain that translocates to the nucleus to induce differentiation 

or apoptosis. Upon activation and cleavage, ErbB4 can also form a complex with TAB2 and N-CoR to repress gene expression. 

Signaling through ErbB networks is modulated through dense positive and negative feedback and feed forward loops, including transcription-independent early loops and late 

loops mediated by newly synthesized proteins and miRNAs. For example, activated receptors can be switched “off” through dephosphorylation, receptor ubiquitination, or 

removal of active receptors from the cell surface through endosomal sorting and lysosomal degradation. 


Arteaga, C.L. and Engelman, J.A. (2014) Cancer Cell 25, 282–303. • Avraham, R. and Yarden, Y. (2011) Nat. Rev. Mol. Cell Biol. 12, 104–117. • Baselga, J. and Swain, 

S.M. (2009) Nat. Rev. Cancer 9, 463–475. • Ferrer-Soler, L., Vazquez-Martin, A., Brunet, J., Menendez, J.A., De Llorens, R., and Colomer, R. (2007) Int. J. Mol. Med. 20, 

3–10. • Moasser, M.M. (2007) Oncogene 26, 6469–6487. • Tebbutt, N., Pedersen, M.W. and Terrance, G.J. (2014) Nat. Rev. Cancer 13, 663–673. • Yarden, Y. and 

Pines, G. (2012) Nat. Rev. Cancer 12, 553–563. • Yarden, Y. and Shilo, B.Z. (2007) Cell 131, 1018. 

Retinoic Acid-Induced Gene 1 (RAIG1) is 

expressed in lung carcinoma and MKN-45 cells. 

RAIG1 (D4S7D) XP ® Rabbit mAb 

#12968: IHC analysis of paraffin-embedded 

human lung carcinoma using #12968 

in the presence of control peptide (A) or 

antigen-specific peptide (B). Confocal 

IF analysis of MKN-45 (C) and 293T 

(D) cells using #12968 (green). Actin 

filaments were labeled with DyLight ® 554 

Phalloidin #13054. Blue pseudocolor = 


A 

C 

B 

D 

© 2004–2015 Cell Signaling Technology, Inc. • We would like to thank Dr. Jinyan Du, Merrimack Pharmaceuticals Inc., Cambridge, MA, for reviewing this diagram. 


www.cellsignal.com/cstgprotein 75


GPR50, an orphan 

GPCR that inhibits 

melatonin receptor 

activity, is expressed 

in 293T but not 

HeLa cells. 

Physiological Roles of GPCRs 

GPCRs are involved in a wide variety of physiological processes, including visual sensing (photoisomerization 

of electromagnetic radiation), taste (release of gustducin in response to bitter- and sweet-tasting 

substances), sense of smell (odorants and pheromones), behavioral and mood regulation (bound 

neurotransmitters: serotonin, dopamine, GABA and glutamate), regulation of the immune system and 

inflammatory response (involvement of chemokine receptors), autonomic nervous system transmission 

(both sympathetic and parasympathetic systems respond to bound ligand), cell density sensing, homeostasis 

modulation (water balance), and involvement in growth and metastasis of some tumor types. It is 

not surprising then that GPCRs are involved in many diseases, and are a significant drug target. 

GPR50 (D1D6I) Rabbit mAb #14032: 

Confocal IF analysis of 293T (left, positive), 

or HeLa (right, negative) cells using 


labeled with DyLight 554 Phalloidin 

#13054 (red). Blue pseudocolor = 


GPCR Structure and Signaling 

GPCRs are characterized by an extracellular N-terminus, followed by seven transmembrane α-helices 

connected by three intracellular and three extracellular loops, and finally an intracellular C-terminus. 

Upon binding a ligand, the seven transmembrane helices of the GPCR arrange themselves into a 

tertiary structure resembling a barrel, forming a cavity within the plasma membrane, thereby exposing 

the external ligand-binding domain (EL-2), though ligands may also bind elsewhere, as is the case for 

bulky ligands for the metabotropic receptors. 

GPCRs are known to exist in a conformational equilibrium between active and inactive biological states. 

Three types of ligands exist: agonists, which shift the equilibrium in favor of active states; inverse 

agonists, which shift the equilibrium in the favor of inactive states; and neutral antagonists, which don’t 

affect the equilibrium. Agonist binding creates a conformational change in the receptor that activates 

an associated G protein (trimer of α, β, and γ subunits, known as Gα, Gβ, and Gγ, respectively), causing 

the G protein to detach from the receptor and separate into GTP-bound Gα and a Gβ/γ dimer. G protein 

activation initiates signaling pathways that regulate cell proliferation, apoptosis, and cytoskeletal 

rearrangements—see details in the signaling pathway to follow. Termination of GPCR signaling occurs 

through phosphorylation by GCPR kinases (GRKs) and binding of β-arrestin proteins, which leads to 

clathrin-mediated receptor internalization and degradation or recycling. 

Commonly Studied GPCR Targets 

Target M P 

AKAP1 

• 

AKAP5 

• 

β1-Adrenergic Receptor 

• 

β2-Adrenergic Receptor 

• 

β-Arrestin 1 

• 

Phospho-β-Arrestin 1 (Ser412) 

• 

β-Arrestin 1/2 

• 

β-Arrestin 2 

• 

Arrestin 1/S-Arrestin 

• 

Gα (pan) 

• 

Gα (z) 

• 


Li, M. et al. (2014) T-cell Immunoglobulin and ITIM Domain 

(TIGIT) Receptor/Poliovirus Receptor (PVR) Ligand Engagement 

Suppresses Interferon-gamma Production of Natural 

Killer Cells via beta-Arrestin 2-mediated Negative Signaling. 

J. Biol. Chem. 289, 17647–17657. 

Seitz, K. et al. (2014) beta-Arrestin interacts with the beta/ 

gamma subunits of trimeric G-proteins and dishevelled in 

the Wnt/Ca(2+) pathway in xenopus gastrulation. PLoS One 

29, e87132. 

Coggins, N.L. et al. (2014) CXCR7 Controls Competition 

for Recruitment of beta-Arrestin 2 in Cells Expressing Both 

CXCR4 and CXCR7. PLoS One 4, e98328. 

Fraisier, C. et al. (2014) Kinetic analysis of mouse brain 

proteome alterations following Chikungunya virus infection 

before and after appearance of clinical symptoms. PLoS One 

9, e91397. 

Kriz, V., et al. (2014) beta-arrestin promotes Wnt-induced 

low density lipoprotein receptor-related protein 6 (Lrp6) 

phosphorylation via increased membrane recruitment of 

Amer1 protein. J. Biol. Chem. 289, 1128–1141. 

Target M P 

Gα (o) 

Gα (i) 

GNB3 

mGluR1 

mGluR2 

GABA(B)R1 

GABA(B)R2 

Phospho-μ-Opioid Receptor (Ser375) 

RAIG1 

RGS4 

• 

• 

• 

• • 

• 

• 

• • 

• 

• • 

• 

Thathiah, A. et al. (2013) beta-arrestin 2 regulates Abeta 

generation and gamma-secretase activity in Alzheimer’s 

disease. Nat. Med. 19, 43–49. 

Stephenson, J.R. et al. (2013) Brain-specific angiogenesis 

inhibitor-1 signaling, regulation, and enrichment in the 

postsynaptic density. J. Biol. Chem. 288, 22248–22256. 

Erickson, C.E. et al. (2013) The beta-blocker Nebivolol Is a 

GRK/beta-arrestin biased agonist. PLoS One 8, e71980. 

Nelson, C.D. et al. (2013) Gpr3 stimulates Abeta production 

via interactions with APP and beta-arrestin2. PLoS One 8, 

e74680. 

Salomonnson, E. et al. (2013) Imaging CXCL12-CXCR4 

signaling in ovarian cancer therapy. PLoS One 8, e51500. 

Canals, M. et al. (2012) Ubiquitination of CXCR7 controls 

receptor trafficking. PLoS One 7, e34192. 

Cintra, D.E. et al. (2012) Unsaturated fatty acids revert 

diet-induced hypothalamic inflammation in obesity. PLoS 

One. 7, e30571. 



nodes within GPCR signaling pathways 

and are commonly studied in GPCR 

research. Primary antibodies, antibody 

conjugates, and antibody sampler kits 

containing these targets are available 

from CST. 





12 


CST antibodies for β-arrestin 1/2 have 

been cited over 12 times in high-impact, 



Arrestin proteins 

function as negative 

regulators of GPCR 

signaling and are 

widely expressed in 

many cell lines. 

A 

kDa 

100 

80 

60 

50 

40 

30 

20 

1 2 3 

β-Arrestin 1 

B 

kDa 

140 

100 

80 

60 

50 

40 

30 

20 

1 2 3 4 5 

β-Arrestin 2 

β-Arrestin 1 (D8O3J) Rabbit mAb 

#12697: WB analysis of extracts from 

MDA-MB-435, Hep G2, and PANC-1 

cells using #12697 (A). 

β-Arrestin 2 (C16D9) Rabbit mAb 


various cell lines using #3857 (B). 

Lanes 

1. MDA-MB-435 

2. Hep G2 

3. PANC-1 

Lanes 

1. HeLa 

2. NIH/3T3 

3. A10 

4. COS 

5. Jurkat 


Audet, M. and Bouvier, M. (2012) Cell 151, 14−23. • Irannejad, R. and von Zastrow, M. (2014) Curr. Opin. Cell Biol. 27, 

109−116. • Kang, D.S., Tian, X., and Benovic, J.L. (2014) Curr. Opin. Cell Biol. 27, 63−71. • O’Hayre, M., Degese, M.S., and 

Gutkind, J.S. (2014) Curr. Opin. Cell Biol. 27, 126−135. • Zhou, L. and Bohn, L.M. (2014) Curr. Opin. Cell Biol. 27, 102−108. 


www.cellsignal.com/cstgprotein 

77



G Protein-Coupled Receptor Signaling: Overview 

RhoA 

β-Arrestin 

AP2 

clathrin 

Receptor 

Internalization 

β-Arrestin 

PI3K 

Akt 

Examples 

Src 

Ras 

Raf 

Erk 

All GPCRs 

p90RSK 

Bad 

M1, H1, 

V2R, OXTR, 

AT 1 R, PAR 

Gα q 

GDP 

Gα q 

GTP 

β,γ 

β,γ 

GRK 

GPR55, SDF-1, 

PAR, S1P 

Gα 12/13 β,γ 

GDP 

Gα 12/13 

GTP 

β,γ 

β1-Adrenergic R 

Wnt Ligand/Frizzled 

Gα s 

GDP 

Gα s 

GTP 

β,γ 

β,γ 

CXCR4, 

GABA-B, LPA 

Gα i β,γ 

GDP 

Gα i β,γ 

GTP 

MMPs 

VEGFR, PDGFR, 

EGFR 

Transactivation 

Src 

PI3K 

Ion 

Channels 

Stat3 

PLCβ PKC Rac RhoA Axin AC 

CDC42 PI3K PLCβ 

Ras 

[cAMP] 

CDC42 

IP3 

PAK 

Akt 

Raf 

PKA 

DAG 

ROCK LATS1/2 GSK-3β 

Ca 2+ 

SOS 

MLK WASP 

PAK 

PKC 

JNK p38 β-catenin CREB 

GSK-3 Erk 

Ras 

Glycogen and 

Actin 

Fatty Acid 

JNK 

Remodeling 

Synthesis 

LATS1/2 

Raf 

Bad mTOR IKK 

Survival Protein 

Bcl-2 

Synthesis 

YAP/TAZ 

p53 

Inactive 

NF-κB 

RTK 

G Protein-Coupled Receptor Signaling to MAP Kinase/Erk 

Gi-Coupled 

Receptor 

Dynamin 

G i 

βγ 

PLCβ 

β-Arrestin GRK2 

Src 

PI3Kγ 

Receptor 


endosome 

H+ 

JNK3 

Focal Adhesions 

Catecholamines 

MP 

Src-like 

Src c-Raf 

β-Arrestin1 MEK 

Erk 

C-TAK1 

TH 

PKC 

RTKs 

IMP 

Src 

GRB2 

RasGEF 

SOS 

Ras 

GAP 

MEK1/2 

KSR 

Src 

FAK 

Gα 

PLCβ q 

PYK2 

GTP 

RACK1 

IP 3 

[Ca 2+ ] 

PKC 

Ras 

c-Raf 

Erk1/2 

Integrins 

Ras 

GRP 

CaMKII,-IV 

Ras 

GRF 

c-Raf B-Raf 

Heterodimer 

Gq-Coupled 

Receptor 

Syn 

GAP 

RGS 

EPAC 

Rap1 

B-Raf 

DUSP6 

cPLA 2 

Synapsins 

AC 

Gα s 

GTP 

[cAMP] 

PKA 

Gs-Coupled 

Receptor 

RGS 

Bcl-xL 

Erk 

YAP/TAZ 

Cytoplasm 

Erk1/2 p90RSK 

PEA-15 

p90RSK 

cdc25 

c-Myc 

p53 

c-Fos 

c-Jun 

YAP/TAZ 

G protein-coupled receptors (GPCRs), also known as 7-transmembrane receptors (7-TMR), form the largest family of cell membrane receptors with over 800 members. 

GPCRs transmit extracellular signals initiated by ligands such as neurotransmitters, hormones, chemokines, and various lipid mediators to the cell interior through coupling to 

heterotrimeric G proteins. G proteins consist of a GDP-bound Gα subunit complexed to β and γ subunits. Upon ligand binding, the α subunit exchanges GDP for GTP, causing 

Gα activation, dissociation from the β/γ subunits, and initiation of signaling pathways that regulate numerous cellular responses, including proliferation, apoptosis, and cytoskeletal 

rearrangements. There are four classes of Gα proteins, each with a unique signaling profile: Gαs, Gαi, Gαq, and Gα12/13. Gαs-coupled receptors activate adenylate 

cyclase, generating cAMP and signaling through PKA . Gαi-coupled receptors inhibit AC and activate CDC42 and PI3K/Akt. Gαq signals through phospholipase C β (PLCβ), 

Rac, and RhoA to regulate cell proliferation and survival by MAPK pathways and actin remodeling via Rock. Gα12/13 activates RhoA, leading to cytoskeletal rearrangements 

as well as activation of the JNK, p38, and Hippo pathways. The β/γ subunits, particularly those associated with Gαi, can initiate signaling independent of Gα proteins, resulting 

in regulation of Akt, PLCβ, and various ion channels. In certain cell types, GPCRs can transactivate receptor tyrosine kinases (RTKs) such as EGFR, PDGFR, and VEGFR, 

thereby activating classical pathways of cell proliferation and survival. Termination of GPCR signaling occurs through phosphorylation by GPCR kinases (GRKs) and binding of 

β-arrestin proteins, which leads to clathrin-mediated receptor internalization and degradation or recycling. β-arrestins can also propagate signals independent of Gα proteins, 

initiating MAPK/Erk, Akt, and RhoA pathways. 


Audet, M. and Bouvier, M. (2012) Cell 151, 14−23. • Entschladen, F., Zänker, K.S., and Powe, D.G. (2011) Cell Cycle 10, 1086−1091. • Kahn, S.M., Sleno, R., Gora, S. 

et al. (2013) Pharmacol. Rev. 65, 545–577. • Lappano, R. and Maggiolini, M. (2012) Acta. Pharmacol. Sin. 33, 351−362. • Lappano, R. and Maggiolini, M. (2011) Nat. 

Rev. Drug Discov. 10, 47−60. • O’Havre, M., Degese, M.S., and Gutkind, J.S. (2014) Curr. Opin. Cell Biol. 27C, 126−135. • Raiagopal, S., Raiagopal, K., and Lefkowitz, 

R.J. (2010) Nat. Rev. Drug Discov. 9, 373−386. • Shukla, A.K., Xiao, K., and Lefkowitz, R.J. (2011) Trends Biochem. Sci. 36, 457−469. • Smrcka, A.V. (2008) Cell. Mol. 

Life Sci. 65, 2191−2214. • Yu, F.X. and Guan, K.L. (2013) Genes Dev. 27, 355−371. 

CREB 

NF-κB 

Erk 

Nucleus 

Nucleus 

p90RSK 

Transcription 

Erk1/2 

FoxO3 

Tumorigenesis 

MSK1/2 

MAPKAPK2 

Progression 

of Cell Cycle 

G protein-coupled receptors (GPCRs) are activated by a wide variety of external stimuli. Upon receptor activation, the G protein exchanges GDP for GTP, causing the dissociation 

of the GTP-bound α and β/γ subunits and triggering diverse signaling cascades. Receptors coupled to different heterotrimeric G protein subtypes can utilize different scaffolds 

to activate the small G protein/ MAPK cascade, employing at least three different classes of Tyr kinases. Src family kinases are recruited following activation of PI3Kγ by β/γ 

subunits. They are also recruited by receptor internalization, crossactivation of receptor Tyr kinases, or by signaling through an integrin scaffold involving Pyk2 and/or FAK. 

GPCRs can also employ PLCβ to mediate activation of PKC and CaMKII, which can have either stimulatory or inhibitory consequences for the downstream MAPK pathway. 


Aoki, Y., Niihori, T., Narumi, Y., Kure, S., and Matsubara, Y. (2008) Hum. Mutat. 29, 992–1006. • Caunt, C.J., Finch, A.R., Sedgley, K.R., and McArdle, C.A. (2006) Trends 

Endocrinol. Metab. 17, 276–283. • Goldsmith, Z.G. and Dhanasekaran, D.N. (2007) Oncogene 26, 3122–3142. • Kim, E.K. and Choi, E.J. (2010) Biochim. Biophys. Acta. 

1802, 396–405. • McKay, M.M. and Morrison, D.K. (2007) Oncogene 26, 3113–3121. 

© 2014–2015 Cell Signaling Technology, Inc. • We would like to thank Dr. Jonathan Violin, Trevena Inc., King of Prussia, PA for reviewing this diagram. 






Activators and 

Inhibitors of PKC 

#9841 Bisindolylmaleimide I, 

Hydrochloride 

Potent inhibitor of 

PKC family members 

#11916 Chelerythrine Chloride 

Cell-permeable inhibitor of 

PKC; activates SAPK/JNK 

and p38-MAPK; induces 

apoptosis in some cell lines 

#12060 Gö6976 

Potent inhibitor of 

calcium-dependent 

PKC family members 

#9995 Ionomycin, Calcium Salt 

Calcium ionophore; 

activates calcium-/calmodulin-dependent 

kinase and 

calcium-dependent PKCs 

#9953 Staurosporine 

Very broad, ATP-competitive 

protein kinase inhbitor (PKC, 

PKA, Src, CaM kinase, etc.) 

#4174 TPA (12-O-Tetradecanoylphorbol-13-Acetate) 

Cell permeable, potent 

activator of PKC; also used 

to activate MAPK. 

Calcium, cAMP, and Lipid Signaling 

Calcium is a critical regulator of a diverse set of cellular functions, and maintaining calcium homeostasis 

is a highly regulated mechanism involving numerous proteins and hormones. Calcium ion channels and 

pumps regulate calcium entry and exit from cells in response to a stimulus. Inside the cell, calcium binding 

proteins regulate local calcium concentrations and help transduce calcium signals. Enzymes such as 

PKC and PLC respond to elevated levels of calcium and transmit signals to downstream signaling nodes. 

Protein Kinase C (PKC) 

Protein kinase C (PKC) family members regulate numerous cellular responses including gene expression, 

protein secretion, cell proliferation, and the inflammatory response. The basic protein structure 

includes an N-terminal regulatory region connected to a C-terminal kinase domain by a hinge region. 

PKC enzymes contain an auto-inhibitory pseudosubstrate domain that binds a catalytic domain 

sequence to inhibit kinase activity. Differences among PKC regulatory regions allow for variable second 

messenger binding and are the basis for the division of the PKC family into 3 broad groups. Conventional 

PKC enzymes (cPKC; isoforms PKCα, PKCβ, and PKCγ) contain functional C1 and C2 regulatory 

domains; cPKC enzyme activation requires binding of diacylglycerol (DAG) and a phospholipid to the 

C1 domain, and calcium binding to the C2 domain. Novel PKC enzymes (nPKC; isoforms PKCδ, PKCε, 

PKCη, and PKCθ) also require DAG binding for activation but contain a novel C2 domain that does not 

act as a calcium sensor. Distantly related protein kinase D proteins are often associated with novel PKC 

enzymes as they respond to DAG but not calcium stimulation. Atypical enzymes (aPKC; isoforms PKCζ 

and PKCι/λ) contain a nonfunctional C1 domain and lack a C2 domain, requiring no second messenger 

binding for aPKC activation. 

The enzyme PDK1 or a close relative is responsible for PKC activation. Control of PKC activity is regulated 

through three distinct phosphorylation events. Phosphorylation occurs in vivo at Thr500 in the 

activation loop, at Thr641 through autophosphorylation, and at the C-terminal hydrophobic site Ser660. 

Phospholipase C (PLC) 

Phosphoinositide-specific phospholipase C (PLC) plays a significant role in transmembrane signaling. 

In response to extracellular stimuli such as hormones, growth factors, and neurotransmitters, PLC 

hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to generate the secondary messengers inositol 

1,4,5-triphosphate (IP3) and diacylglycerol (DAG). At least four families of PLCs have been identified: 

PLCβ, PLCγ, PLCδ and PLCε. PLC activity is largely regulated by phosphorylation. For example, 

phosphorylation of PLCβ3 at Ser1105 by PKA or PKC inhibits activity, whereas phosphorylation of PLCγ 

at Tyr 771, 783, and 1245 by both receptor (EGFR) and nonreceptor tyrosine kinases (Syk) results 

in activation. In addition, members of the PLCβ subfamily are activated by the α- or β/γ-subunits of 

heterotrimeric G-proteins and play an important role in GPCR signaling cascades. 

Growth factor stimulation results in phosphorylation of PLCγ1 at Tyr783. 

A 

Phospho-PLCγ1 (Tyr783) (D6M9S) Rabbit mAb #14008: Confocal IF analysis of A-431 cells, untreated (A), treated with hEGF #8916 

(100 ng/ml, 5 min) (B), or treated with hEGF #8916 (100 ng/ml, 5 min) and λ phosphatase (C), using #14008 (green). Blue pseudocolor 

= DRAQ5 ® #4084 (fluorescent DNA dye). WB analysis of extracts from NIH/3T3 cells, untreated (-) or stimulated with hPDGF-BB #8912 

(5 min; +), and from A-431 cells, untreated (-) or stimulated with hEGF #8916 (5 min; +) (D), using #14008 (upper) and β-Actin (D6A8) 


B 

C 

D 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

60 

50 

40 

30 

1 2 

– + – – 

– – – + 

Phospho- 

PLCγ1 

(Tyr783) 

β-Actin 

hPDGF-BB 

hEGF 

Akt/PKB 

p70S6K 

PDK1 

PKCζ/λ PKCδ PKCθ PKCµ PKCα/βII 


Insulin, etc. 

PI3K 

Activated 

By TPA 

Receptor 

Calcium Binding Proteins 

Calcium is a critical second messenger for intracellular signaling pathways that regulate a diverse 

range of biological functions such as cell growth, motility, contractility, membrane trafficking, neurotransmitter 

release, apoptosis, and differentiation. Low molecular weight calcium binding proteins 

such as calmodulin, calbindin, S-100, paravalbumin, and troponin C bind to calcium via EF hand 

domains and help transduce intracellular signals by activating downstream target proteins or act as 

calcium buffering proteins to regulate local calcium concentrations. For example, the calcium binding 

protein calbindin is expressed in discrete neuronal populations within the CNS, including Purkinje cells, 

and is thought to act as an intracellular calcium buffering protein. Calbindin is highly expressed in 

neurons during migration and differentiation. 

MARCKs 

Calcium binding protein calbindin is expressed 

in discrete neuronal populations within the CNS. 

Phorbol ester TPA 

activates PKCδ, resulting 


at Thr505. 

kDa 

105 

76 

57 

Phospho- 

PKCδ 

(Thr505) 

0 15 30 60 120 240 TPA (min) 

Phospho-PKCδ (Thr505) Antibody 


U-937 cells, untreated or TPA-treated 

(0.2 µM), using #9374. 

PKCθ is a novel protein kinase C predominantly expressed in T cells. 

CD3 

10 4 

10 3 

10 2 

10 1 

10 0 10 1 10 2 10 3 10 4 

10 0 

10 0 10 1 10 2 10 3 10 4 

Rabbit Isotype Control 

PKCθ 

PKCθ (E1I7Y) Rabbit mAb 

#13643: Flow cytometric analysis 

of mouse splenocytes using Rabbit 

(DA1E) mAb IgG XP ® Isotype Control 

#3900 (left) and #13643 (right). 

Splenocytes were co-stained with 

anti-CD3 APC and the Anti-rabbit 

IgG (H+L), F(ab’) 2 Fragment (PE 

Conjugate) #8885 was used as a 

secondary antibody. 

Calbindin (D1I4Q) XP ® Rabbit mAb 

#13176: Confocal IF analysis of normal rat 

brain (A) using #13176 (green) and GFAP 

(GA5) Mouse mAb #3670 (red). Blue pseudocolor 

= DRAQ5 ® #4084 (fluorescent DNA 

dye). IHC analysis of paraffin-embedded rat 

brain (B) using #13176. 

A 

B 


www.cellsignal.com/cstcalcium 81


Calcium pump 

protein ATP2A2/ 

SERCA2 is widely 

expressed in 


Calcium Channels and Pumps 

Maintaining proper calcium concentrations within the cell is critical for effective cell signaling and 

requires a variety of channels and pumps to transport calcium ions across intracellular and plasma 

membranes. Ion channels move calcium ions into the cell or out from intracellular storage compartments 

(with the gradient), effectively raising cytoplasmic calcium concentrations. The three types of 

calcium ion channels are broadly classified by their ability to open in response to a ligand, second 

messenger, or membrane potential (voltage-dependent calcium channels; VDCC). For example, the IP3 

receptor requires the second messenger inositol 1,4,5-triphosphate (IP3) for activation and is located 

on the endoplasmic reticulum (ER) where it regulates release of intracellular calcium stores. 

As cytoplasmic calcium concentrations rise, calcium can be transported back outside the cell or into 

storage within the sarcoplasmic reticulum or ER by calcium pumps. Calcium pump proteins are calcium- 

ATPases that use the energy of ATP hydrolysis to retrotransport calcium across plasma or ER membranes, 

thus maintaining the calcium gradient necessary for rapid signaling. For example, the calcium 

pump ATP2A2/SERCA2 is responsible for regulating calcium transport across sarcoplasmic reticulum and 

ER membranes, and its activity can be regulated through a variety of post-translational modifications. 

ATP2A2/SERCA2 Antibody #4388: WB analysis 


Lanes 

1. Hep G2 

2. RD 

3. C2C12 

4. Jurkat 

5. NIH/3T3 

6. PC-12 

IP3 receptor, a calcium ion channel activated by 

second messengers, is expressed in brain tissue. 

IP3 Receptor 1 (D53A5) Rabbit mAb #8568: WB analysis of extracts from mouse and 

rat brain using #8568. 


kDa 

200 

140 

100 

80 

60 

50 

40 

kDa 

200 

140 

100 

1 2 

IP3 

Receptor 1 

Lanes 

1. mouse brain 

2. rat brain 

Mitochondrial Calcium Uniporter 

The mitochondrial calcium uniporter (MCU) is a calcium channel specifically located within the 

mitochondrial inner membrane. Mitochondrial calcium uniporter regulator 1 (MCUR1) is a multi-pass, 

transmembrane protein that directly interacts with MCU and plays an essential role in the regulation of 

calcium uptake and maintenance of mitochondrial calcium homeostasis. Regulation of MCU by MCUR1 

may be critical for a variety of cellular functions, including signal transduction, bioenergetics, and cell 

death and survival. 

Mitochondrial calcium uniporter 

is expressed in many cell lines. 

MCUR1 Antibody #13706: WB analysis of extracts from 

various tissues and cell lines using #13706 (upper) and 

β-Actin (D6A8) Rabbit mAb #8457 (lower). 

1 2 3 4 5 6 

kDa 

60 

50 

40 

30 

20 

50 

40 

ATP2A2/ 

SERCA2 

1 2 3 4 5 6 

MCUR1 

β-Actin 

Lanes 

1. mouse kidney 

2. mouse testis 

3. human kidney 

4. rat kidney 

5. C6 

6. Neuro-2a 


Bublitz, M., Musgaard, M., Poulsen, H., et al. (2013) J. Biol. Chem. 288, 10759–10765. • Freeley, M., Kelleher, D., Long, A. 

(2011) Cell. Signal. 23, 753–762. • Newton, A.C. (2010) Am. J. Physiol. Endocrinol. Metab. 298, 395–402. • Patron, M., 

Raffaello, A., Granatiero, V. et al. (2013) J. Biol. Chem. 288, 10750–10758. • Rossi, A.M., Tovey, S.C., Rahman, T., et al. 

(2012) Biochim. Biophys. Acta. 1820, 1214–1227. • Yáñez, M., Gil-Longo, J., and Campos-Toimil, M. (2012) Adv. Exp. Med. 

Biol. 740, 461–482. • Yang, Y.R., Follo, M.Y., Cocco, L., and Suh, P.G. (2013) Adv. Biol. Regul. 53, 232–241. 

Commonly Studied Calcium, cAMP, and Lipid Signaling Targets 

Target M P S Target M P S Target M P S 

β1-Adrenergic Receptor • PDE5 

• Phospho-Phospholamban 

(Ser16/Thr17) 

• 

AKAP1 • 

PIP4K2A • 

AKAP5 • 

PIP4K2B • PLCβ3 • • 

Annexin A1 • • PIP5K1A • Phospho-PLCβ3 • 

(Ser537) 

Annexin A2 • 

PIP5K1C • 

Phospho-PLCβ3 

Annexin A7 • PKA C-α • • • 

• 

(Ser1105) 

ApoA1 • 

Phospho-PKA C-α • • 

PLCγ1 • • 

ApoA4 • 

(Thr197) 

Phospho-PLCγ1 

ApoA5 • 

PKA RI-α/β • 

• • 

(Tyr783) 

ApoM • 

Phospho-PKC (pan) • 

Phospho-PLCγ1 • • 

(βII Ser660) 

ASM 

• 

(Ser1248) 

Phospho-PKC (pan) 

ATP2A1/SERCA1 • • 

• 

PLCγ2 

• 

(γ Thr514) 

ATP2A2/SERCA2 • • 


Phospho-PKC (pan) • 

• 

Pan-Calcineurin A • 

(Tyr759) 

(ζ Thr410) 

Calmodulin • 


PKCα 

• 

• 

(Tyr1217) 

Calumenin • 

Phospho-PKCα/β II • 

PLD1 

CBARA1/MICU1 • 

• 

(Thr638/641) 

Phospho-PLD1 (Thr147) 

CFTR 

• PKCδ • • 

• 

Phospho-PLD1 (Ser561) 

Choline Kinase α • 

Phospho-PKCδ (Tyr311) • 

• 

PLD2 

cPLA2 • • Phospho-PKCδ (Thr505) • 

• 

PRK2 

Phospho-cPLA2 • Phospho-PKCδ/θ • 

• • 

(Ser505) 

(Ser643/676) 

PKA RI-α • 

Cyclic AMP • 

PKCε • • RyR1 • 

DAG Lipase β • 

PKCθ • • S100A1 • 

Gα (pan) • Phospho-PKCθ (Thr538) • S100A4 • • 

Gα (z) 

• PKCζ • • • S100A6 • 

Gα (i) 

• Phospho-PKCζ/λ • S100A10 • 

Gα (o) 

• 

(Thr410/403) 

S100B • 

Gelsolin • • • 

PKD/PKCµ • S100P 

• • 

INPP4b • • 

Phospho-PKD/PKCµ • nSMase1 • 

(Ser744/748) 

IP3 Receptor • • 

SPHK1 • • 

Phospho-PKD/PKCµ 

Phospho-IP3 Receptor • • 

• STIM1 • • 

(Ser916) 

(Ser1756) 

STIM2 

• 

PKD2 

MARCKS • 

• • 

TGM2 • 

PKD3/PKCν 

Phospho-MARCKS • • 

• 

TRPV3 

• 

(Ser152/156) 

Phospho-PRK1 • 

TSPO 

• 

(Thr774)/PRK2 (Thr816) 

Phospho-MARCKS • 

WFS1 

(Ser167/Ser170) 

Phospholamban • 

• • 

NIPSNAP1 • 


Volk, L. et al. (2013) PKM-zeta is not required for hippocampal 

synaptic plasticity, learning and memory. Nature 

493, 420–423. 

Paul, S. et al. (2014) T cell receptor signals to NF-kappaB 

are transmitted by a cytosolic p62-Bcl10-Malt1-IKK signalosome. 

Sci. Signal. 7, ra45. 

Dusaban, S.S. et al. (2013) Phospholipase C epsilon links 

G protein-coupled receptor activation to inflammatory 

astrocytic responses. Proc. Natl. Acad. Sci. USA 110, 

3609–3614. 

Xiang, S.Y. et al. (2013) PLCepsilon, PKD1, and SSH1L 

transduce RhoA signaling to protect mitochondria from 

oxidative stress in the heart. Sci. Signal. 6, ra108. 

Varsano, T. et al. (2013) Inhibition of melanoma growth by 

small molecules that promote the mitochondrial localization 

of ATF2. Clin. Cancer Res. 19, 2710–2722. 

Ke, G. et al. (2013) MiR-181a confers resistance of cervical 

cancer to radiation therapy through targeting the proapoptotic 

PRKCD gene. Oncogene 32, 3019–3027. 

Stumpf, C.R. et al. (2013) The translational landscape of the 

mammalian cell cycle. Mol. Cell. 52, 574–582. 

Gobbi, G. et al. (2013) Proplatelet generation in the mouse 

requires PKCepsilon-dependent RhoA inhibition. Blood 122, 

1305–1311. 

Tuszynski, M.H. et al. (2012) Concepts and methods for 

the study of axonal regeneration in the CNS. Neuron 7, 

777–791. 

Qu, Y. et al. (2012) Phosphorylation of NLRC4 is critical for 

inflammasome activation. Nature 490, 539–542. 



nodes within calcium, cAMP, and lipid 

signaling pathways and are commonly 

studied in calcium, cAMP, and lipid 

signaling research. Primary antibodies, 









58 


CST antibodies for PKC have been 

cited over 58 times in high-impact, peerreviewed 



www.cellsignal.com/cstcalcium 83

Signalin 

Signalin 

Wnt1 

Wnt5A 

LRP5/6 

mut 

BMPRI 

BMPs 

BMPRII 

mut 

exp 

mut 

ligand 

GPCR 

GPCR 

GPCR [IP 3 ] 

[DAG] 

PLCβ 

PLCβ 

PAR-1 

p120 

Ras 

CKI 

CKI 

Dsh 

PAR-1 

SARA 

TGFβ RI 

TGFβ 

mut 

GPCR 

mut, trans, amp, exp 

mut 

Ras 

mut 

mut 

mut 

mut 

mut 

mut 

Src 

mut 

α-catenin 

ERK 

VEGF 

EPO 

PDGF 

AC 

PI3Kγ 

Ubiquitin 

mut 

mut 

14-3-3 

KIBRA 

[Ca 2+ ] 

PI3K 

Smad7 

Smad7 

Crk 

C3G 

[cAMP] 

EPAC 

CAMKII 

PKC 

PP2A 

GSK-3β 

APC 

WTX 

Axin 

mut 

ALK 

ROS1 

mut 

mut 

mut 

Smad4 

amp, mut, exp 

14-3-3 

YAP 

SOS 

GRB2 

[PIP 3 ] 

Ubiquitin 

Mst1/2 LATS1/2 

SAV1 MOB1 

TAB1 

TAB2 

mut 

TAB1 

TAB2 

mut 

mut 

Rap 

PKA 

RasGRF 

Ras 

NF1 

Raf-1 

B-Raf 

14-3-3 

HPH 

Ras c-Raf 

MEK1/2 

ERK1/2 

MP1 

RasGEF 

Smad4 

Smurf 

NLK 

Smad4 

mut 

MKK3/6 

mut 

Akt 

RAR 

mut 

mut 

SOX 

mut 

E2F 

amp 

mut, del 

mut 

amp, mut, exp 

amp, exp 

CBP 

TCF/LEF 

Integrin 

Src 

FAK 

GRB2 

GRAF 

SOS 

Rap 

RalGEF 

TEAD 

Smad 

mut 

mut 

KSR 

14-3-3 c-Raf 

VHL 

HIF-1β 

mut 

del 

ERK1/2 

Smad4 

p107 

E2F 

mut 

mut 

mut 

CDK7 

RalA 

RalB 

Cdc2 

Wee1A 

exp 

Miz-1 

Smad4 

RalBP1 

Cdc42 Actin 

Rac 

MEK1/2 

MP1 

ERK1/2 

exp 

ERK1/2 

p90 RSK 

HIF-1β 

PAK1 

Cdc25 

B/C 

Myc 

Miz-1 

mut, amp 

mut, amp 

Cytoskeleton 

CREB 

Rho 

JNK 

HPV-E7 

HDAC 

Rb 

DP E2F 

Rad52 

Rad51 

FANC 

D2 

mut 

mut, del 

mut 

vir 

exp 

Abl 

PI3K 

class I 

ULK1 

Beclin 

FIP200 

Atg14 

Atg13 

mut 

p90 RSK 

mut 

amp, trans 

trans 

Elk-1 

NBS1 

BRCA1 

Chk1 

Chk2 

mut 

mut, amp 

mut 

CDK4/6 

Cdc25A 

mut 

mut mut 

mut 

mut,del 

Cdc2 

Cyclin A 

LC3-II 

Atg7/3 

Atg4 

LC3-I 

mut,del 

4E-BP 

S6K 

DP E2F 

MDM2 

p21 

Rb 

amp 

GSK-3 

mut 

mut 

mut 

SHP1 

HIPK2 

ARF 

MDM2 

CDK7 

trans, mut 

(amino acids 

& glucose) 

TSC2 

TSC1 

mTOR 

Raptor 

p21 p27 

FKHR/ 

FOXO 

JAK 

JAK 

STAT 

SHP2 

GRB2 

STAT 

SOS 

Shc 

Ras 

LKB1 

PI3K 

AMPK 

1011010100110101101100101 

Rheb 

Cdc25A 

cyclin E, A 

myc, cdc2 

p107, E2F 

0 10 1 0101 

0 

0 10 1 0101 

0 10 10 10 1 

101010110110110 

HPV-E6 

mut 

amp 

1011010100110101101010101 

trans 

vir 

mut 

amp, exp 

CDK2 

Cyclin A 

mut 

mTOR 

Rictor 

CDK2 

Cyclin E 

Akt 

BRD4 

TET 

SirT1 

DNMT3 

XIAP 

Lamins 

PDK1 

Bax 

Bcl-2 

Casp-2 

p21 

p27 

amp 

mut,del 

amp,exp 

amp, mut, exp 

mut 

amp, trans 

trans 

NOXA 

PUMA 

Smac 

AIF 

PARP 

CAD 

mut 

mut 

cIAP 

RelA 

NFκB2 

RelB 

PP2A 

c-Raf 

HDAC 

Sin3 

PKM2 

pyruvate 

LDHA 

14-3-3 Bim 

Apaf-1 

ICAD 

CAD 

amp, trans 

mut 

mut,del 

IκB 

p53 

Gab1 

PI3K 

RTK 

Gab2 

IRS-1 

Cbl 

PTEN 

PI3K 

mut 

PFK 

mut 

CytC 

IκB 

PDK1 

Ras 

G-6-P 

F-6-P 

Fructose 

Bisphosphate 

amp, trans 

IDH1 

mut 

Su(Fu) 

KIF-7 

Su(Fu) 

KIF-7 

mut, del 

citrate 

IDH2 

Akt 

Bax 

Bcl-2 JNK 

tBid 

ASK 

MKK7 

Bid 

TAB2 

TAB3 

IKKα 

IKKβ 

mut 

trans 

citrate 

MEKK3 

NUMB 

Hardcover 

Paperback 

Glucose 

mut 

FLIPs 

NIK 

Ras 

mut 

amp 

trans 

del 

vir 

exp 

mut 

Dsh 

Presenilin 

IRAK 

TRAF6 

Myd88 

RAIDD 

TRAF2 

mut 

mut 

mut 

mut 

mut 

Amplification 

Translocation 

Deletion 


amp, mut, exp 

from PDB 

oncogene 

Hh 

Fas/DR 

mut 

trans 

Delta 

Jagged 

TACE 

trans 

/ NF- 

N otch g 

S ignalin 

Signalin 



Calcium, cAMP, 

and Lipid Signaling 

Kinase-Disease 

Associations 


PKACα AGC Cancer Multiple mutations of regulatory subunit (PRKAR1A; OMIM:188830) 

cause Carney complex tumors and sporadic adrenal and thyroid 

tumors. Altered PRKAR1A transcript editing associated with lupus. 

PKACa knockout mouse has phenotypes similar to Carney complex 

(Medline:18413734). OMIM:601639. 

PKC 

(multi-isoform) 

AGC CNS Inh Multiple agonists and antagonists of the PKC family have consistent 

effects on inhibiting or activating manic behavior in humans and rats. 

Tamoxifen has secondary effect as PKC inhibitor and shown antimanic 

activity in rat model and a clinical trial (Medline:18316672, 

17641532). 

Pathways in 

PKCα AGC Cancer 


Mut, 

Del, OE, 

Act 

A point mutation is seen in several pituitary and thyroid tumors (Medline:9167945). 

Deleted in a melanoma cell line. Complex expression 

pattern in breast cancer (Medline 15459489, 15454252). Therapeutic 

target in lung, gastric and prostate cancer (Medline:15447994, 

15313921, 15174974). May mediate multidrug resistance (Medline:12390766). 

Mouse models indicate a role in heart contractility 

(Medline:14966518). Inhibitors: LY-900003 (antisense. aka Affinitak/ 

ISIS 3521/aprinocarsen), Safingol, Go6976. OMIM:176960. 

Human Cancer 

The Pathways in Human Cancer Poster summarizes the signaling nodes implicated 

in cancer initiation and progression and features peer-reviewed protein mapping with 

scientifically accurate rendering of the molecular structures of proteins. The poster 

accompanies the text A Biology of Cancer by Robert A. Weinberg (Second Edition). 

PKCβ AGC Autism 

Cancer 

Diabetes 

PKCγ AGC Neurodegeneration 

Pain 

PKCδ AGC Cancer 


CNS 

PKCε AGC Cancer 


CNS 

PKCθ AGC Cancer 

Immunity 

SNP 

Two promoter SNPs associated with diabetic nephropathy 

(Medline:12874455), correlating with induction of renal expression 

by high glucose, reduction in renal function by a specific PKCβ 

inhibitor and successful inhibitor treatment of rodent models of 

diabetic nephropathy (Medline:12955673). PKCβ inhibition has also 

been proposed to treat diabetic retinopathy (Medline:12507628) 

and diabetic vascular complications (Medline:11903393). Ectopic 

expression in mouse heart leads to cardiac hypertrophy. Elevated 

expression is seen in and promotes early stages of colon cancer 

in mouse models (Medline:11245437). May mediate multidrug 

resistance (Medline:12697075). Activation protects astrocytes from 

ischemic injury (Medline:15165841). SNPs associated with autism 

(Medline:16027742). Inhibitors: LY333531 (ruboxistaurin; Phase 3 

for diabetic neuropathy and retinopathy), LY317615 (Eli Lilly: isoform 

selective). OMIM:176970. 

Mut Point mutations linked to dominant spinocerebellar ataxia type 14 

[OMIM:605361]. Knockout and inhibitor studies show role in pain 

perception (Medline:14762097, 9323205). OMIM:176980. 

Expr 

Amp, 

Mut 

Pro-apoptotic. Reduced expression correlated with progression of colon 

and other cancers (Medline:15054085, 12657722, 12591726). 

Inhibition may drive chemo-resistant cancers to apoptosis. Activated 

and promotes apoptosis in cardiac and neuronal cells after ischemicreperfusion 

injury (Medline:14654063, 15295022). Activator: 

bistrane A. Inhibitors: rottlerin, KAI-9803 (KAI; Phase 2 trials for 

reperfusion injury), dV1-1. OMIM:176977. 

Amplified and rearranged in thyroid cancers (Medline:9683604, 

10438519, 11994357). Promotes growth of an androgen-independent 

prostate cancer cell line (Medline:11956106) and transforms 

fibroblasts in culture (Medline:11968018). Activation protects cardiac 

myocytes and neurons from ischemic injury (Medline:15165841) 

and antagonizes PKCδ. Amyloid β peptide inhibits PKCε activity 

and may contribute to the pathogenesis of Alzheimer's disease 

(Medline:15207847). Inhibitor studies indicate role in pain perception 

(Medline:1043272). Multi-isoform inhibitors include: Bryostatin-1 

(BRYO) from Aphios and Perifosine/D-21266 from Baxter while KAI- 

1678 is a selective inhibitor. OMIM:176975. 

Required for activation and proliferation of mature T cells. Proposed 

as a target for T cell leukemias (Medline:12188914). Knockout is 

resistant to fat-induced insulin resistance. OMIM:600448. 

Molecular: Act Activated • Amp Amplified • Del Deleted • Expr Expression • GOF Gain-of-function • Inh Inhibitor 

Studies • LOF Loss-of-function • LOH Loss-of-heterozygosity • Meth Methylation • Model model organism studies • 

Mut Mutation OE Overexpression • SNP Single Nucleotide Polymorphism • Splice Splicing change • Trans Translocation 

Features 

Peer-reviewed protein mapping and accurate molecular rendering of proteins. 

Summarizes the latest signaling nodes implicated in cancer signaling. 

Pathway-specific color-coded nodes. 

Revised Edition 

Angiogenesis 

Wnt Signaling 

H ippo g 


R-spondin 

T GF β g 

R-spondin 

Frizzled 

Frizzled 

Receptor Tyrosine Kinase (RTK) 

E-cadherin 

CD44 

FAT4 

LGR4,5,6 

γ 

β 

α 

γ 

β 

α 

ZNRF3/RNF43 

β-catenin 

γ 

β 

α 

Dsh Akt 

α 12/13 

γ 

β 

α s 

Smad1/5/8 

Smad2/3 

TGFβ RII 

EGFR 

HER2 

FRMD 

Mer 

Smad1/5/8 

Smad2/3 

Ras Signaling 

β-catenin 

TAK 

RasGRP 

RasGAP 

Normoxia 

Hypoxia 

SynGAP 

β-catenin 

YAP/TAZ 

Smad1/5/8 

Smad2/3 

TAK p38 

C 

PI3K ILK 

RIAM/Talin 

RasGAP 

RhoGEFs 

β-catenin 

MKK7 JNK 

HIF-1α(-2α) 

other TFs 

YAP/TAZ 

myc, cyclin D, CD44 

Smad1/5/8 

Smad2/3 

Cyclin H 

Regulated 

Exocytosis 

Exocyst 

Complex 

Atg16/12/5 

Rab8a (c-Mel) 

p90 RSK JNK 

HIF-1α(-2α) 

Fos 

Jun 

myc, cyclin D 

Cyclin B 

p15, p21, p27 

Smad2/3 

DNA Repair 

Myc 

Max 

autophagosome 

Myc 

Max 

cyclin D, myc 

cyclin D, myc 

SQSTM1 

Autophagy 

Fos 

Jun 

phagopore 

membrane 

nucleation 

p15 p16 p18 p19 

UV 

Cyclin D 

DNA Damage 

Cyclin D 

STAT STAT 

(Inactive) (Active) 

IR 

* * 

ATM 

ATR 

Scienstists at Cell Signaling Technology co laborate 

with key opinion leaders in cance research to create 

reference pathway diagrams that reflect the latest 

thinking in the research community. Over 40 pathway 

diagrams cu rently exist, including the pathways 

represented here and many others. 

www.ce lsignal.com/CSTcancer 

DNA- 

PK 

p53 

p53 

Nutrients 

cyclin D 

The Pathways in Human Cancer poster summarize some of the key 

signaling pathways implicated in tumorigenesis and tumor 

progression in humans. Within each pathway, gene products known 

to be mutated in human tumors—oncogenes and tumor suppressor 

genes—are coded with information on types of genetic alterations 

and confe red capabilities to the tumor. Proteins are shown using 

structural representatives. New to this revised poster edition are 

signaling pathways for Hippo Signaling, Autophagy, Warburg E fect, 

and Epigenetic Regulation. 

This poster was created by scientists at Ce l Signaling Technology in 

co laboration with Robert A. Weinberg and others a the forefront of 

cance research. Expanded versions of each pathway, including 

additional downstream signaling nodes, can be found at 

www.ce lsignal.com/CSTcancer. 

R 

Cyclin H 

Cytokine 

Receptor 

Aurora 

A 

Transcription 

MDM2 Bcl-x L 

Bad 

p53 

myc, cyclin D, cyclin E 

cyclin D 

CSL/CBF1 

Gli PKA 

KMT2/MLL 

KDM6A/UTX 

KMT4/DOT1L 

SWI/SNF 

KMT6/EZH2 

NFκB1/2 

CSL/CBF1 

p300 

Epigenetic Signaling 

Ce l Signaling Technology would like to thank 

digizyme for their co laboration on the design 

and concept of this poster. Please visit 

www.digizyme.com to see more of their work. 

NICD 

[PIP 3 ] 

lactate 

Casp-9 

Casp-3,6,7 

HAT HES1 

Akt Signaling 

NICD 

Gli 

[PIP 3 ] 

Hexokinase 

Krebs 

Cycle 

Bak TRADD 

IKKα IKKβ 

NFκB1/2 

RelA 

Gli 

Fatty acid 

synthesis 

TAK 

NEMO 

Direct stimulatory modification 

Direct inhibitory modification 

Multistep stimulatory modification 

Tentative stimulatory modification 

Transcriptional contribution 

TYPES OF GENETIC ALTERATIONS: 

Point mutation 

Increased expression 

(unknown mechanism) 

TYPES OF CONFERRED CAPABILITIES: 

Evading apoptosis 

Self-su ficiency in growth signals 

Insensitivity to anti-growth signals 

Tissue invasion & metastasis 

Limitless replicative potential 

Sustained angiogenesis 

∞ 

transporter 

Ras Common protein name 

Structural representative 

tumor suppressor 

Type of genetic alteration 

Type of confe red capability 


FADD 

Casp-8,10 

RIP TRADD 

TRAF 

2/6 

TRAF 

3 

Notch 

Furin 

Fringe 

NIC 

Cleaved 

Notch 

CDO 

BOC 

Ptch 

Smo 

Ptch 

Smo 

IL1R 

TNFR 

TNFR 

CDO 

BOC 

D eath Receptor κB g 

This poster accompanies the textbook The Biology of Cancer, Second Edition 

by Robert A. Weinberg and published by Garland Science. 

Dr. Weinberg is a founding member of the Whitehead Institute for Biomedical 

Research and the Daniel K. Ludwig Professor of Cancer Research a the 

Massachuse ts Institute of Technology. 

ISBN 978-0-8153-4219-9 

ISBN 978-0-8153-4220-5 www.garlandscience.com 

Signalin 

H edgehog g 

Pathways in 

Human Cancer 

Poster 

To request free copies of this 

poster please visit our website. 

www.cellsignal.com/phcposter 

Cancer 

Research 


Please visit our website to learn 

more about the scientific tools and 

educational resources we have online 

for cancer signaling and proteomic 

analysis, including discussion of key 

disease drivers. 

www.cellsignal.com/cancerguide 

85

03 


kDa 

60 

50 

40 

30 

20 

60 

50 

40 

30 

20 

10 

– 

Raji 

+ 

MCF7 

– + 

Phospho- 

Bim 

(Ser77) 

Cell Growth and Death 

Apoptosis 

Apoptosis is a tightly controlled process of cell death characterized by nuclear condensation, cell 

shrinkage, membrane blebbing, and DNA fragmentation. The central regulators of apoptosis are the 

caspases, a family of cysteine proteases that fall into two broad categories. The “initiator” caspases-2, 

-8, -9, -10, and -12 are closely coupled to upstream, pro-apoptotic signals and act by cleaving and 

activating the downstream “executioner” caspases-3, -6, and -7 that modify proteins ultimately responsible 

for programmed cell death. Targets such as PARP, lamin A/C, α-/β-actin, GAS2, α-fodrin, acinus, 

and many others are cleaved by executioner caspases and serve as markers of apoptosis. There are 

two primary apoptotic pathways that lead to caspase activation: the intrinsic and extrinsic pathways. 

Apoptosis-inducing agent, staurosporine, results in cleavage/activation 

of caspase-3 and morphological characteristics of cell death. 

Cleaved Caspase-3 (Asp175) 

(D3E9) Rabbit mAb (Alexa Fluor ® 

647 Conjugate) #9602: Confocal IF 

analysis of HeLa cells, untreated (left) 

or treated with Staurosporine #9953 

(1 μM, 4 hr; right), using #9602 (blue 

pseudocolor). Actin filaments were labeled 

with Alexa Fluor ® 488 Phalloidin 

#8878 (green). Red = Propidium Iodide 

(PI)/RNase Staining Solution #4087. 

Etoposide treatment results in cleavage of PARP, a marker for apoptosis. 

Etoposide #2200: WB analysis of extracts from Jurkat cells, untreated (-) or etoposide-treated 

(25 μM, overnight; +), using Cleaved PARP (Asp214) (D64E10) XP ® Rabbit mAb #5625 (upper) 

or total PARP Antibody #9542 (lower). 

Intrinsic Apoptosis 

The intrinsic apoptosis pathway is activated by cell stress, DNA damage, developmental cues, and 

withdrawal of survival factors. This pathway is regulated by the Bcl-2 family of proteins, which consists 

of 3 classes: the pro-apoptotic effectors (Bax and Bak); the pro-apoptotic BH3-only proteins (Bad, Bid, 

Puma, Bim, Noxa, Bmf, Hrk, and Bik); and the anti-apoptotic proteins (Bcl-2, Bcl-xL, Bcl-w, and Mcl-1, 

A1/Bfl1). The pro-apoptotic effectors localize to the mitochondrial membrane and control mitochondrial 

permeability and release of cytochrome c, AIF, Smac/Diablo, HtrA2, Endo G, and others. The proapoptotic 

effectors are activated by BH3-only proteins either through direct interaction or antagonism of 

anti-apoptotic proteins. Members of the BH3-only class are generally regulated by apoptotic stimuli, 

either at the level of transcription or through phosphorylation or other post-translational modifications. 

The cytochrome c released as a result of Bcl-2 protein action binds to Apaf-1 and forms the apoptosome, 

a large, multiprotein, flower-like structure that binds and activates caspase-9, resulting in 

caspase-3 cleavage and other apoptotic endpoints. 

Cleaved 

PARP 

(Asp214) 


kDa 

140 

100 

80 

60 

50 

140 

100 

80 

60 

50 

PARP 

Cleaved 

PARP 

(Asp214) 

– + Etoposide 

Bim EL 

Bim L 

Bim S 

TPA treatment results in phosphorylation of Bim at Ser77, a 

TPA 

modification known to promote Bim proteasomal degradation. 

Phospho-Bim (Ser77) (D4H12) Rabbit mAb #12433: WB analysis of extracts from Raji and MCF7 cells, untreated (-) 

or treated with TPA #4174 (200 nM, 30 min; +), using #12433 (upper) or Bim (C34C5) Rabbit mAb #2933 (lower). 

Cytochrome c release 

is a key indicator of 

intrinsic apoptosis. 

Extrinsic Apoptosis 

Caspases can also be activated through the extrinsic, or death receptor, pathway. The death receptors 

consist of members of the TNFR family (TNFR1/2, Fas, and DR3/4/5/6) and their associated ligands 

(TNF-α, FasL, TRAIL, TWEAK). Ligand binding induces receptor activation and recruitment of the adaptor 

proteins FADD or TRADD via their death domains (DD). Together, the ligand-bound receptor, adaptor 

protein, and procaspase-8 form the death-inducing signaling complex (DISC), a multiprotein complex 

that activates caspase-8 and initiates downstream apoptotic signaling. Signaling through TNF-family 

cytokines can lead to either apoptosis through DISC or survival by activation of NF-κB and the prosurvival 

genes, Bcl-2 and FLIP. Cycloheximide, which inhibits NF-κB gene regulation, is commonly 

used with TNF-α to induce apoptosis. 

Treatment with TNF-α and cycloheximide results in 

detection of caspase-8 cleavage products, p43 and p18. 

Cleaved Caspase-8 (Asp387) (D5B2) XP ® Rabbit mAb (Mouse Specific) #8592: WB analysis of 

extracts from CTLL-2 cells, untreated or treated with cycloheximide (CHX, 10 μg/ml, overnight) followed 

by hTNF-α #8902 (20 ng/ml, 4 hr), using #8592. 

Necrosis and Necroptosis 

The highly regulated system of apoptotic cell death is not the only way cells can die. “Accidental” 

death, called necrosis, is an unregulated form of cell death characterized by cell swelling, plasma 

membrane rupture, and inflammation. 

kDa 

100 

80 

60 

50 

40 

30 

20 

– + 

chapter 03: Cell Growth and Death 

p43 

p18 

CHX/TNF-α 

Recently, a form of programmed necrosis has also been identified. Activation of the death receptors can 

lead to caspase-independent cell death termed necroptosis. Necroptosis is induced by the TNF-family 

of cytokines and mediated by the kinases RIP1 and RIP3. In the absence of caspase-8 activation, RIP1 

forms a complex with the key regulator of necroptosis, RIP3, and its downstream substrate, MLKL. 

Together, these proteins create complex IIb in conjunction with FADD, caspase-8, and cFLIP, forming 

the necroptosome. 

Apoptosis Assays 

There are many assays available for measuring cell death. Apoptosis assays measure key apoptotic 

markers such as activation of caspase-3 or release of cytochrome c. Assays measuring necrosis typically 

focus on the cell permeability that is characteristic of necrotic plasma membrane rupture. Some 

assays, such as Annexin V, can differentiate between apoptosis and necrosis using a dual marker 

system—annexin V binding to membrane-bound phosphatidylserine indicates early apoptosis whereas 

propidium iodide labeling identifies the leaky cell membranes associated with necrosis. 


Dickens, L.S., Powley, I.R., and Hughes, M.A., et al. (2012) Exp. Cell Res. 318, 1269–1277. • Favaloro, B., Allocati, N., and 

Graziano, V., et al. (2012) Aging 4, 330–349. • Giampietri, C., Starace, D., Petrungaro, S., et al. (2014) Int. J. Cell Biol. 49027. 

• McIlwain, D.R., Berger, T., and Mak, T.W. (2013) Cold Spring Harb. Perspect. Biol. 5, a008656. • Moriwaki, K. and Chan, 

F.K. (2013) Genes Dev. 27, 1640−1649. • Shamas-Din, A., Kale, J., and Leber B., et al. (2013) Cold Spring Harb. Perspect. 

Biol. 5, a008714. 

Cytochrome c (6H2.B4) Mouse 


HeLa cells, untreated (left) or treated 

with Staurosporine (1 μM) #9953 and 

Z-VAD (50 μM) for 3 hr (right), using 

#12963 (green). Blue pseudocolor= 


Assay Kits for 

Measuring Cell 

Death & Viability 

Annexin V-FITC Early Apoptosis 

Detection Kit #6592 

Differentiates between early apoptosis 

and necrosis through annexin 

V and propidium iodide labeling 

Caspase-3 Activity 

Assay Kit #5723 

Measures caspase-3 activity 

through cleavage of fluorescently 

labeled caspase-3 substrate 

Resazurin Cell Viability 

Kit #11884 

Measures metabolic activity as 

an indicator of cell viability using 

fluorescent detection 

XTT Cell Viability Kit #9095 

Measures metabolic activity as 

an indicator of cell viability using 

colorimetric detection 

Cytochrome c Release 

Flow Kit #13648 

Identifies cytochrome c release by 

flow cytometry using a cytochrome 

c antibody 

www.cellsignal.com/cstapoptosis 87




nodes within apoptotic signaling 


in apoptosis research. Primary 


antibody sampler kits containing 









Inducers of Apoptosis 

Stimulating apoptosis with chemical modulators or cytokines is a common strategy for investigating effects on downstream 

signaling. These agents induce apoptosis by a variety of methods, typically through inhibition of key cellular processes or kinases. 

#9972 Brefeldin A Interferes with ER to Golgi transport; typically used to study vesicle trafficking but 

prolonged exposure causes apoptosis 

#2112 Cycloheximide Inhibits protein synthesis and is often used in conjunction with TNF-α to induce cell death 

#2200 Etoposide Inhibits Topoisomerase II 

#9807 Paclitaxel Binds β-tubulin and prevents microtubule depolymerization 

#9885 Roscovitine Inhibits CDK1, CDK2, CDK5 to cause cell cycle arrest and apoptosis 

#9953 Staurosporine Potently inhibits PKC; also inhibits PKA, PKG, CAMKII, and MLCK 

#5927 Doxorubicin Inhibits DNA and RNA synthesis through intercalating the DNA double helix 

#8902 Human Tumor Necrosis 

Factor-α (hTNF-α) 

#5178 Mouse Tumor Necrosis 

Factor-α (mTNF-α) 

#4698 Mouse His 6 Tumor Necrosis 

Factor-α (m His 6TNF-α) 

Commonly Studied Apoptosis Targets 


A1/Bfl-1 

• 

APR3 

• 

Acinus 

• 

AIF • • • 

Alix 

• 

AP-2α 

• • 

AP-2β 

• 

AP-2g 

• 

Phospho-AP2M1 (Thr156) • 

Apaf-1 • • • 

Aven 

• 

Bad 

• • • • 

Phospho-Bad (Ser112) • • • • 

Phospho-Bad (Ser136) • • 

Phospho-Bad (Ser155) • • 

Bak1 • • • 

BAP31 

• 

Bax • • • 

Bcl-2 

• • • • 

Phospho-Bcl-2 (Thr56) • 

Phospho-Bcl-2 (Ser70) • • • 

Bcl-w 

• 

Bcl-xL • • • • 

BCL2L10 

• 

A1/Bfl-1 

• 

BID 

• • 

Bik 

• 

Bim • • • • 

Phospho-Bim (Ser55) 

• 

Phospho-Bim (Ser69) • • 

Phospho-Bim (Ser77) • 

BIRC6 

• 

Bit1 

• 

Bmf 

• 

c-IAP1 

• • 

Triggers extrinsic apoptosis and/or survival through death receptor signaling 



#5452 Fas Ligand Triggers extrinsic apoptosis through death receptor signaling 


c-IAP2 

• 

Caspase Cleavage Motif • 

Caspase-1 

• 

Cleaved Caspase-1 (Asp297) • 

Caspase-2 

• 

Caspase-3 • • • 

Cleaved Caspase-3 (Asp175) • • • • 

Caspase-4 

• 

Caspase-5 

• 

Caspase-6 

• 


Caspase-7 

• • 

Cleaved Caspase-7 (Asp198) • • 

Caspase-8 

• 




Caspase-9 

• • 




Non-Phospho-Caspase-9 (Ser196) 

• 

Caspase-11 

• 

Caspase-12 

• 

Caspase-14 

• 

Cytochrome c • • • 

DAP1 

• 

DAPK1 

• 

DAPK3/ZIPK 

• 

Daxx 

• 

Cleaved Drosophila Dcp-1 (Asp216) • 

DcR1 

• 

DcR2 

• 

DcR3 

• 

DFF45/DFF35 

• 


Cleaved DFF45 (Asp224) • 

DIDO1 

• 

DR3 

• 

DR5 

• • 

Phospho-DR6 (Ser562) • 

DRAK2 

• 

eIF4G2/p97 

• 

Endonuclease G 

• 

FADD 

• 

Phospho-FADD (Ser194) • 

FAF1 

• 

FAIM 

• 

Fas 

• 

Fas Ligand 

• 

FLIP • • 

α-Fodrin 

• 

Cleaved α-Fodrin (Asp1185) • 

Granzyme A 

• 

Granzyme B 

• 

HIPK2 

• 

HtrA2/Omi • • • 

Cleaved Drosophila ICE (drICE) 

(Asp230) 

• 

Lamin A/C • • • 

Cleaved Lamin A/C (Small Subunit) • • 

Phospho-Lamin A/C (Ser22) • • • 

Lamin B1 

• 

Lamin B2 

• 

LAP2α 

• 

Livin 

• 

Mad-1 

• 

Maspin 

• 

Max 

• 

Mcl-1 • • • 

Phospho-Mcl-1 (Ser64) • 

Phospho-Mcl-1 (Ser159/Thr163) • 

Phospho-Mcl-1 (Thr183)/ 

MST2(Thr180) 

Phospho-MLKL 

Mst1 

• 

• 

• 


Mst2 

• 

Mst3 

• 

Mst4 

• 

c-Myc • • • 

Phospho-c-Myc (Ser62) • 

N-Myc 

• 

PAR-4 

• 

Phospho-PAR-4 (Thr163) • 

PARP1 • • 

Cleaved-PARP1 (Asp214) • • • • 

PDCD4 

• 

PEA-15 • • • 

Phospho-PEA-15 (Ser104) • 

Perforin 

• 

PHLDA3 

• 

Puma • • • 

RIP1 

• • 

RIP3 

• • 

Siva-1 

• 

Smac/Diablo 

• 

Survivin 

• • • • • 

Phospho-Survivin (Thr34) • • 

TANK 

• 

TAX1BP1 

• 

TMS1 

• 

TP/ECGF1 

• • 

TNF-R2 

• 

TRADD 

• • 

TRAF1 

• 

TRAF2 

• 

Phospho-TRAF2 (Ser11) • 

TRAF3 

• 

TRAF6 

• 

TRAIL 

• 

VDAC1 

• • 

VDAC2 

• 

WWOX 

• 

XAF1 

• • 

XIAP • • • 

1,240 


CST antibodies for cleaved caspase-3 

(Asp175) have been cited over 1,240 

times in high-impact, peer-reviewed 

publications from the global research 

community. 


Bjordal, M. et al. (2014) Sensing 

of amino acids in a dopaminergic 

circuitry promotes rejection of an 

incomplete diet in Drosophila. Cell 

156, 510–521. 

Doitsh, G. et al. (2014) Cell death by 

pyroptosis drives CD4 T-cell depletion 

in HIV-1 infection. Nature 505, 

509–514. 

Walczynski, J. et al. (2014) Sensitisation 

of c-MYC-induced B-lymphoma 

cells to apoptosis by ATF2. Oncogene 

33, 1027–1036. 

de Poot, S.A. et al. (2014) Granzyme 

M targets topoisomerase II alpha to 

trigger cell cycle arrest and caspasedependent 

apoptosis. Cell Death 

Differ. 21, 416–426. 

Pencheva, N. et al. (2014) Broadspectrum 

therapeutic suppression of 

metastatic melanoma through nuclear 

hormone receptor activation. Cell 

156, 986–1001. 

Ardestani, A. et al. (2014) MST1 is a 

key regulator of beta cell apoptosis 

and dysfunction in diabetes. Nat. 

Med. 20, 385–397. 

von Figura, G. et al. (2014) The 

chromatin regulator Brg1 suppresses 

formation of intraductal papillary 

mucinous neoplasm and pancreatic 

ductal adenocarcinoma. Nat. Cell Biol. 

16, 255–267. 

Beug, S.T. et al. (2014) Smac 

mimetics and innate immune stimuli 

synergize to promote tumor death. 


Gao, T. et al. (2014) Pdx1 maintains 

beta cell identity and function by 

repressing an alpha cell program. Cell 

Metab. 259–271. 

Chen, Y. et al. (2014) Hyperactivation 

of mammalian target of rapamycin 

complex 1 (mTORC1) promotes 

breast cancer progression through 

enhancing glucose starvation-induced 

autophagy and Akt signaling. J. Biol. 

Chem. 289, 1164–1173. 

Razorenova, O.V. et al. (2014) The 

apoptosis repressor with a CARD domain 

(ARC) gene is a direct hypoxiainducible 

factor 1 target gene and 

promotes survival and proliferation of 

VHL-deficient renal cancer cells. Mol. 

Cell. Biol. 34, 739–751. 


www.cellsignal.com/cstapoptosis 89

c-IAP 

TRADD 

FADD 



Regulation of Apoptosis Overview 

Inhibition of Apoptosis 

TNF, FasL, TRAIL 

TNF, FasL, TRAIL 

Trophic Factors 

TNF-α 

TNF-R1 

Survival Factors: 


Cytokines, etc. 

Cytoplasm 

IKKα 

IκBα 

TAK1 

IKKγ 

IκBα 

NF-κB 

NF-κB 

Cytoplasm 

Nucleus 

IKKβ 

Calpain 

[Ca 2+ ] 

FLIP 

XIAP 

c-IAP 

TRAF2 

NIK Casp-8,-10 

RIP 

TRAF2 

Casp-12 

Casp-9 

ER Stress 

RIP1 

TRADD 

NF-κB 

FADD 

CYLD 

PARP 

Itch 

FLIP 

TRADD 

FADD 

BID 

ROCK 

• Cell shrinking 

• Membrane 

blebbing 

DFF40 

XIAP 

AIF 

RIP RAIDD 

PIDD 

Casp-2 

p53 

SirT2 

HtrA2 

Arts 

Casp-3,-6,-7 

Lamin 

A/C 

APP 

DNA 

Fragmentation 

RIP 

Smac/ 

Diablo 

Endo G 

TRAF2 

tBID 

DFF40 

JNK 

Cyto c 

DFF45 

ASK1 

Bax 

DNA Damage 

Bak 

Mcl-1 

Casp-9 

Apaf-1 

AIF 

PKC 

Bcl-2 

Bim 

ATM/ATR 

p53 

Puma 

Noxa 

p90RSK 

Puma 

Bcl-xL 

Noxa 

HECTH9 

Endo G 

Chk1/2 

FoxO1 

p53 

Bad 

p53 

PI3K 

Cell Cycle 

Erk1/2 

cdc2 

14-3-3 

Akt 

Cellular Stress 

FoxO1 

JNK 

JNK 

Bim 

c-Jun 

NIK 

IKKγ CDC37 

IKKβ HSP90 

IκB 

IκB 

NF-κB 

NF-κB 

Cytoplasm 

Nucleus 

NF-κB 

cIAP 

TAK1 

TAB 

IKKα IKKβ 

IKKγ 

TRAF2 

TRADD 

HSP90 

Casp-8 

XIAP 

CYLD 

HSP70 

FADD 

HSP90 

JNK 

HSP27 

A20 

GSK-3 

FoxO1 

PTEN 

PI3K 

PIP 3 

Akt 

Bcl-2 

Casp-3,-6,-7 

Bax 

HSP27 

Apoptosis 

Casp-9 

Apaf-1 Cyto c HECTH9 HSP70 

Akt 

FLIP 

XIAP 

A20 

FLIP 

BID 

Smac/ 

Diablo 

RIP1 

HSP70 

tBID 

HSP27 

HSP90 

Bax 

Bax 

Mcl-1 

Bak 

Bcl-xL 

Bim 

Bim 

PDK1 

p70S6K 

FAS 

Bim 

Bad 

Bim 

PKA 

[cAMP] 

FoxO1 

Ras 

Raf 

Erk1/2 

FoxO1 

PKC 

p90RSK 

Jak 

Src 

CREB Stat1 Stat3 

Bcl-2 

Bcl-xL 

Cytoplasm 

CREB 

Stat1 

Stat3 

HSP90 

Apoptosis is a regulated cellular suicide mechanism characterized by nuclear condensation, cell shrinkage, membrane blebbing, and DNA fragmentation. Caspases, a family 

of cysteine proteases, are the central regulators of apoptosis. Initiator caspases (including caspase-2, -8, -9, -10, -11, and -12) are closely coupled to pro-apoptotic signals. 

Once activated, these caspases cleave and activate downstream effector caspases (including caspase-3, -6, and -7), which in turn execute apoptosis by cleaving cellular 

proteins following specific Asp residues. Activation of Fas and TNFR by FasL and TNF, respectively, leads to the activation of caspase-8 and -10. DNA damage induces the 

expression of PIDD, which binds to RAIDD and caspase-2 and leads to the activation of caspase-2. Cytochrome c released from damaged mitochondria is coupled to the 

activation of caspase-9. XIAP inhibits caspase-3, -7, and -9. Mitochondria release multiple pro-apoptotic molecules, such as Smac/Diablo, AIF, HtrA2, and Endo G, in addition 

to cytochrome c. Smac/Diablo binds to XIAP, preventing it from inhibiting caspases. Caspase-11 is induced and activated by pathological pro-inflammatory and pro-apoptotic 

stimuli and leads to the activation of caspase-1, thereby promoting inflammatory response and apoptosis by directly processing caspase-3. Caspase-12 and caspase-7 are 

activated under ER stress conditions. Anti-apoptotic ligands, including growth factors and cytokines, activate Akt and p90RSK. Akt inhibits Bad by direct phosphorylation and 

prevents the expression of Bim by phosphorylating and inhibiting the Forkhead family of transcription factors (FoxO). FoxO promotes apoptosis by upregulating pro-apoptotic 

molecules such as FasL and Bim. 


Degterev, A. and Yuan J. (2008) Nat. Rev. Mol. Cell Biol. 9, 378–390. • Fuchs, Y. and Steller H. (2011) Cell 147, 742–758. • Indran, I.R., Tufo, G., Pervaiz, S., and Brenner 

C. (2011) Biochim. Biophys. Acta. 1807, 735–745. • Kaufmann, T., Strasser, A., and Jost, P.J. (2012) Cell Death Differ. 19, 42–50. • Kurokawa, M. and Kornbluth, S. 

(2009) Cell 138, 838–854. • Pradelli, L.A., Bénéteau, M., and Ricci, J.E. (2010) Cell. Mol. Life Sci. 67, 1589–1597. • Van Herreweghe, F., Festjens, N., Declercq, W., and 

Vandenabeele, P. (2010) Cell. Mol. Life Sci. 67, 1567–1579. 

Cell survival requires the active inhibition of apoptosis, which is accomplished by inhibiting the expression of pro-apoptotic factors as well as promoting the expression of 

anti-apoptotic factors. The PI3K pathway, activated by many survival factors, leads to the activation of Akt, an important player in survival signaling. PTEN negatively regulates 

the PI3K/Akt pathway. Activated Akt phosphorylates and inhibits the pro-apoptotic Bcl-2 family members Bad, Bax, caspase-9, GSK-3, and FoxO1. Many growth factors and 

cytokines induce anti-apoptotic Bcl-2 family members. The Jaks and Src phosphorylate and activate Stat3, which in turn induces the expression of Bcl-xL and Bcl-2. Erk1/2 

and PKC activate p90RSK, which activates CREB and induces the expression of Bcl-xL and Bcl-2. These Bcl-2 family members protect the integrity of mitochondria, preventing 

cytochrome c release and the subsequent activation of caspase-9. TNF-α may activate both pro-apoptotic and anti-apoptotic pathways; TNF-α can induce apoptosis by 

activating caspase-8 and -10, but can also inhibit apoptosis via NF-κB, which induces the expression of anti-apoptotic genes such as Bcl-2. cIAP1/2 inhibit TNF-α signaling 

by binding to TRAF2. FLIP inhibits the activation of caspase-8. 


Brumatti, G., Salmanidis, M., and Ekert, P.G. (2010) Cell. Mol. Life Sci. 67, 1619–1630. • Fuchs, Y. and Steller, H. (2011) Cell 147, 742–758. • Fulda, S. and Vucic, D. 

(2012) Nat. Rev. Drug Discov. 11, 109–124. • Kaufmann, T., Strasser, A., and Jost, P.J. (2012) Cell Death Differ. 19, 42–50. • Lopez, J. and Meier, P. (2010) Curr. Opin. 

Cell Biol. 22, 872–881. • Rong, Y. and Distelhorst, C.W. (2008) Annu. Rev. Physiol. 70, 73–91. • Srinivasula, S.M. and Ashwell, J.D. (2008) Mol. Cell 30, 123–135. • 

Zhang, X., Tang, N., Hadden, T.J., and Rishi, A.K. (2011) Biochim. Biophys. Acta. 1813, 1978–1986. 

© 2002–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Junying Yuan, Harvard Medical School, Boston, MA, for reviewing this diagram. 






Death Receptor Signaling 

Mitochondrial Control of Apoptosis 

MKK1 

JNR 

ASK1 

Bcl-2 

Casp-9 

Casp-6 

FasL 

Daxx 

Fas/ 

CD95 

tBID 

Bcl-2 

RIP 

BID 

Apaf1 

XIAP 

FADD 

Casp-8, -10 

FLIP 

Cyto c 

TNF-α 

TNF-R1 

TRAF2 

TRADD 

cIAP1/2 

Casp-8 

FADD 

Necroptosis 

RIP1 

Complex 

IIb 

RIP3 

MLKL 

CYLD 

RIP1 

XIAP 

IKKα/β/γ 

Casp-8 

FADD 

RIP1 

Casp-3 

Lamin A Actin GAS2 α-Fodrin ROCK1 DFF45 Acinus PARP 

ub 

Smac 

Complex 

IIa 

Active 

Caspase-8 

DFF40 

TNF-α 

TNF-R2 

TRAF2 TRAF1 

cIAP1/2 

IκB 

NF-κB 

TRAF3 

TRAF2 

TRAF1 

Casp-7 

RIP 

cIAP1/2 

APO-3L/ 

TWEAK 

DR3 

APO-3 

TRADD TRADD 

IκB 

p65/ 

RelA 

FADD 

Casp-8,-10 

NF-κB 

p50 

FADD 

p52 

APO-2L/ 

TRAIL 

DR4/5 

NF-κB 

p100 

NIK 

IKKα 

RelB 

RelB 

Processing 

Survival Factors: 

Growth Factors, Cytokines, etc. 

PKA 

14-3-3 

PKC 

Erk1/2 

p90RSK 

Bad 

14-3-3 

Bad 

Cytosolic 

Sequestration 

HSP60 

Casp-3 

PI3K 

Akt 

p70 S6K 

Calcineurin 

Apaf-1 

Casp-9 

ub 

XIAP 

Arts 

FADD 

BID 

FasL 

Fas/ 

CD95 

Casp-8,-10 

tBID 

Bax 

Bak 

Bad 

Cyto c 

HECTH9 

Bcl-xL 

HtrA2 

Mcl-1 

Bcl-xL 

tBID 

Smac/ 

Diablo 

Mcl-1 

Bim 

LC8 

Microtubules 

DLC2 

Bmf 

Puma 

AIF 

Bim 

Bcl-2 

HECTH9 

Endo G 

Bcl-2 

Bmf 

Bcl-2 

Noxa 

Death Stimuli: 

Survival Factor Withdrawal 

Hrk 

Bcl-2 

Bax 

Bak 

p53 

ATM/ 

ATR 

JNK 

Hrk 

ING2 

[NAD] 

SirT2 

JNK 

Bax 

Casp-2 

ARD1 

NatA 

Acetyl-CoA 

RAIDD 

CaMKII 

PIDD 

Cell Shrinkage 

Membrane Blebbing 

DNA 

Fragmentation 

Apoptosis 

Chromatin 

Condensation 

Transcription 

Survival 

Pro-Apoptotic 

Pro-Survival 

Apoptosis 

DNA Damage 

Genotoxic Stress 

Apoptosis can be induced through the activation of death receptors including Fas, TNFαR, DR3, DR4, and DR5 by their respective ligands. Death receptor ligands characteristically 

initiate signaling via receptor oligomerization, which in turn results in the recruitment of specialized adaptor proteins and activation of caspase cascades. Binding of FasL 

induces Fas trimerization, which recruits initiator caspase-8 via the adaptor protein FADD. Caspase-8 then oligomerizes and is activated via autocatalysis. Activated caspase-8 

stimulates apoptosis via two parallel cascades: it can directly cleave and activate caspase-3, or alternatively, it can cleave Bid, a pro-apoptotic Bcl-2 family protein. Truncated 

Bid (tBid) translocates to mitochondria, inducing cytochrome c release, which sequentially activates caspase-9 and -3. TNF-α and DR-3L can deliver pro- or anti-apoptotic 

signals. TNFαR and DR3 promote apoptosis via the adaptor proteins TRADD/FADD and the activation of caspase-8. Interaction of TNF-α with TNFαR may activate the NF-κB 

pathway via NIK/IKK. The activation of NF-κB induces the expression of pro-survival genes including Bcl-2 and FLIP, the latter can directly inhibit the activation of caspase-8. 

FasL and TNF-α may also activate JNK via ASK1/MKK7. Activation of JNK may lead to the inhibition of Bcl-2 by phosphorylation. In the absence of caspase activation, stimulation 

of death receptors can lead to the activation of an alternative programmed cell death pathway termed necroptosis by forming complex IIb. 


Declercq, W., Vanden Berghe, T., and Vandenabeele, P. (2009) Cell 138, 229–232. • Fuchs, Y. and Steller H. (2011) Cell 147, 742–758. • Kantari, C. and Walczak, H. 

(2011) Biochim. Biophys. Acta. 1813, 558–563. • Kaufmann, T., Strasser, A., and Jost, P.J. (2012) Cell Death Differ. 19, 42–50. • Lavrik, I.N. and Krammer, P.H. (2012) 

Cell Death Differ. 19, 36–41. • Van Herreweghe, F., Festjens, N., Declercq, W., and Vandenabeele, P. (2010) Cell. Mol. Life Sci. 67, 1567–1579. • Wajant, H. and 

Scheurich, P. (2011) FEBS J. 278, 862–876. 

The Bcl-2 family of proteins regulate apoptosis by controlling mitochondrial permeability. The anti-apoptotic proteins Bcl-2 and Bcl-xL reside in the outer mitochondrial wall 

and inhibit cytochrome c release. The pro-apoptotic Bcl-2 proteins Bad, Bid, Bax, and Bim may reside in the cytosol but translocate to mitochondria following death signaling, 

where they promote the release of cytochrome c. Bad translocates to mitochondria and forms a pro-apoptotic complex with Bcl-xL. This translocation is inhibited by survival 

factors that induce the phosphorylation of Bad, leading to its cytosolic sequestration. Cytosolic Bid is cleaved by caspase-8 following signaling through Fas; its active fragment 

(tBid) translocates to mitochondria. Bax and Bim translocate to mitochondria in response to death stimuli, including survival factor withdrawal. Activated following DNA damage, 

p53 induces the transcription of Bax, Noxa, and Puma. Upon release from mitochondria, cytochrome c binds to Apaf-1 and forms an activation complex with caspase-9. 

Although the mechanism(s) regulating mitochondrial permeability and the release of cytochrome c during apoptosis are not fully understood, Bcl-xL, Bcl-2, and Bax may influence 

the voltage-dependent anion channel (VDAC), which may play a role in regulating cytochrome c release. Mule/ARF-BP1 is a DNA damage-activated E3 ubiquitin ligase 

for p53, and Mcl-1, an anti-apoptotic member of Bcl-2. 


Brenner, D. and Mak, T.W. (2009) Curr. Opin. Cell Biol. 21, 871–877. • Chalah, A., Khosravi-Far, R. (2008) Adv. Exp. Med. Biol. 615, 25–45. • Lindsay, J., Esposti, M.D., 

and Gilmore, A.P. (2011) Biochim. Biophys. Acta. 1813, 532–539. • Ola, M.S., Nawaz, M., and Ahsan, H. (2011) Mol. Cell. Biochem. 351, 41–58. • Pradelli, L.A., 

Bénéteau, M., and Ricci, J.E. (2010) Cell. Mol. Life Sci. 67, 1589–1597. • Rong, Y. and Distelhorst, C.W. (2008) Annu. Rev. Physiol. 70, 73–91. • Speidel, D. (2010) 

Trends Cell Biol. 20, 14–24. • Suen, D.F., Norris, K.L., and Youle, R.J. (2008) Genes Dev. 22, 1577–1590. 






Nutrient deprivation 

results in accumulation 

of Atg13 at sites 

of autophagy. 

Autophagy 

Autophagy is a dynamic cellular recycling system that degrades cytoplasmic contents, abnormal 

protein aggregates, and excess or damaged organelles so that the building blocks, such as amino 

acids, can be used to create new cellular components. Autophagy occurs when the protein, organelle, 

or cytoplasmic content to be degraded is surrounded by a small portion of membrane, creating 

an autophagosome. The autophagosome is then fused to the lysosome, creating an autolysosome 

and resulting in degradation of cellular components via lysosomal enzymes. Autophagy is generally 

activated by conditions of nutrient deprivation but has also been associated with physiological as well 

as pathological processes such as development, differentiation, neurodegenerative diseases, stress, 

infection, obesity, and cancer. 

Autophagy Induction 

The molecular machinery of autophagy was largely discovered in yeast and is directed by a number 

of autophagy-related (Atg) genes. The mammalian serine/threonine kinase ULK (similar to yeast Atg1) 

plays a critical role in autophagy induction, acting as a convergence point for upstream signals. Activation 

of mTOR through Akt or MAPK signaling results in inhibition of ULK and suppression of autophagy, 

whereas negative regulation of mTOR through p53 and AMPK signaling relieves this inhibition and 

promotes autophagy. In addition, AMPK also phosphorylates and activates ULK directly. ULK forms a 

complex with Atg13 and the scaffold protein FIP200. 

Signaling through ULK1 activates phosphoinositide 3-kinase class III (PI3K class III or hVps34), a lipid 

kinase that produces phosphatidylinositol-3-phosphate (PIP 3 ) and forms a large complex with associated 

proteins p105/Vsp15, Beclin-1, UVRAG, Atg14, and Rubicon. As autophagy signaling is induced, 

membranes from cytoplasmic vesicles collect and fuse, forming the phagophore. The PI3K class III 

complex acts upon phagophore lipids, creating binding sites for autophagic proteins and initiating the 

formation of autophagosomes. 

Treatment with AMPK activators result 

in phosphorylation of ULK1 at Ser555. 

Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb #5869: WB analysis of 

extracts from MCF7 cells, untreated or treated with Oligomycin #9996 

(0.5 μM, 30 min), and C2C12 cells, untreated or treated with hydrogen 

peroxide (10 mM, 5 min), using #5869. 

PI3 Kinase class III, which is a lipid kinase 

that initiates autophagosome formation, is 

expressed in mouse brain and C6 cells. 

PI3 Kinase Class III (D9A5) Rabbit mAb #4263: WB analysis of extracts 

from mouse brain and C6 cells using #4263. 


kDa 

200 

140 

100 

80 

60 

kDa 

200 

140 

100 

80 

60 

MCF7 

C2C12 

– + – – 

– – – + 

1 2 

Phospho- 

ULK1 

(Ser555) 

Oligomycin 

Hydrogen Peroxide 

Atg13 (E1Y9V) Rabbit mAb #13468: 

Confocal IF analysis of PANC-1 cells, 

untreated (left) or nutrient-starved 

with Earle’s balanced salt solution for 

3 hr (right), using #13468 (green). 



PI3 Kinase 

Class III 

Lanes 


2. C6 

Autophagosome Formation 

Formation of the autophagosome requires a pair of essential ubiquitin-like conjugation systems, Atg12- 

Atg5 and LC3-phosphatidylethanolamine (LC3-PE). In the first conjugation reaction, Atg12 is covalently 

bound to Atg5 by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10. The Atg12-Atg5 

conjugate then forms a complex with Atg16L1 and localizes to phagophore membranes. In the 

second conjugation step, LC3 is first primed for lipidation through cleavage of its C-terminus by Atg4, 

generating LC3-I. LC3-I is then conjugated to PE in a ubiquitin-like reaction that requires Atg7 and 

Atg3 (E1- and E2-like enzymes, respectively). The lipidated form of LC3, known as LC3-II, is attached 

to the autophagosome membrane. Throughout this process, the phagophore membrane continues to 

expand and surround cytoplasmic components that have been designated for degradation. Sequestosome 

1 (SQSTM1, p62), a ubiquitin binding protein that binds LC3, facilitates this process by delivering 

SQSTM1-containing protein aggregates to the autophagosome for destruction. 

Nutrient starvation and chloroquine treatment result in 

expression of LC3A/B in human and mouse cells. 

HeLa (human) 

C2C12 (mouse) 

Mitophagy 

Mitophagy is a selective autophagic process specifically designed for the removal of damaged or unneeded 

mitochondria from a cell. Polyubiquitination of mitochondrial membrane proteins by Parkin results 

in the recruitment of autophagy adaptor proteins that bind to LC3 via their LC3-interacting region 

(LIR). A second level of ubiquitin-independent regulation occurs through the mitochondrial membrane 

proteins BNIP3 and BNIP3L/NIX, which also contain LIRs and directly recruit autophagic machinery to 

induce autophagosome formation in certain cell types. 

BNIP3L/Nix (D4R4B) Rabbit mAb 

#12396: Confocal IF analysis of A172 

cells, untreated (left) or cobalt chloridetreated 

(100 μM, overnight; right), using 

#12396 (green). Blue pseudocolor = 


A B C 


Alers, S., Löffler, A.S., Wesselborg, S., and Stork, B. (2012) Mol. Cell. Biol. 32, 2–11. • Codogno, P., Mehrpour, M., and 

Proikas-Cezanne, T. (2012) Nat. Rev. Mol. Cell Biol. 13, 7–12. • Ding, W.X. and Yin, X.M. (2012) Biol. Chem. 393, 547–564. 

• Feng, D., Liu, L., Zhu, Y., and Chen, Q. (2013) Exp. Cell. Res. 319, 1697–1705. • Jin, M. and Klionsky, D.J. (2014) FEBS 

Lett. 588, 2457–2463. • Schneider, J.L. and Cuervo, A.M. (2014) Curr. Opin. Genet. Dev. 26C, 16–23. • Papinski, D. and 

Kraft, C. (2014) Autophagy 10, 1338–1340. 


Atg5, which regulates 

the first step in autophagosome 

formation, 

is widely expressed. 

kDa 

140 

100 

80 

60 

50 

40 

30 

20 

1 2 3 4 5 6 

Atg5 

Atg5 (D5G3) Rabbit mAb #9980: WB 

analysis of extracts from various cell lines 

using #9980. 

Lanes 

1. PANC-1 4. ACHN 

2. K-562 5. HT-1080 

3. SH-SY5Y 6. COS-7 

LC3A/B (D3U4C) XP ® Rabbit mAb 

#12741: Confocal IF analysis of HeLa 

(upper) and C2C12 (lower) cells, 

chloroquine-treated (50 μM, overnight) 

(A), nutrient-starved with EBSS (3 hr) 

(B) or untreated (C) using #12741 

(green) and β-Actin (13E5) Rabbit mAb 

(Alexa Fluor ® 555 Conjugate) #8046 

(red). Blue pseudocolor= DRAQ5 ® 


Cobalt chloride, 

which induces the 

hypoxia regulator 

HIF-1α, results in 

accumulation of the 

mitophagy regulator 

protein BNIP3L/Nix. 

www.cellsignal.com/cstautophagy 95



nodes within autophagy signaling 


in autophagy research. Primary 









207 


CST antibodies for LC3B have been 




Antibody 

Validation 

Principles 

Please visit our website to learn more 

about what Antibody Validation means 

at Cell Signaling Technology. 

www.cellsignal.com/cstvalidation 

Commonly Studied Autophagy Targets 

Target M P S Target M P S 

Ambra1 

• Phospho-Beclin-1 

Atg3 

(Ser93/96) • 

• 

Atg4A 

Bif-1 

• 

• 

Atg4B 

BNIP3 

• • • 

• 

Atg4C 

BNIP3L/Nix 

• • 

• 

Atg5 

FIP200 

• • • 

• 

Atg7 

GABARAP 

• • • 

• 

Atg9A GABARAPL2 

• • 

• 

Atg12 

LC3A 

• • 

• • 

Atg13 LC3A/B 

• • 

• • 

Atg14 

LC3B 

• • 

• • • 

Atg16L1 

MTMR3 

• 

• 

Atg101 

MTMR14 

• 

• 

Beclin-1 

NBR1 

• • • 

• 

Phospho-Beclin-1 (Ser15) 

Rubicon 

• 

• 

SQSTM1/p62 • • • 


Mancias, J.D. et al. (2014) Quantitative proteomics identifies 

NCOA4 as the cargo receptor mediating ferritinophagy. 

Nature 509, 105–109. 

Bejarano, E. et al. (2014) Connexins modulate autophagosome 

biogenesis. Nat. Cell Biol. 16, 401–414. 

Jang, Y.H. et al. (2014) Phospholipase D-mediated autophagic 

regulation is a potential target for cancer therapy. Cell 

Death Differ. 21, 533–546. 

Kim, J. et al. (2014) Differential regulation of distinct Vps34 

complexes by AMPK in nutrient stress and autophagy. Cell 

152, 290–303. 

Mealer, R.G. et al. (2014) Rhes, a striatal-selective protein 

implicated in Huntington disease, binds beclin-1 and activates 

autophagy. J. Biol. Chem. 289, 3547–3554. 

Efeyan, A. et al. (2014) Regulation of mTORC1 by the Rag 

GTPases is necessary for neonatal autophagy and survival. 

Nature 93, 679–683. 

Brot, S. et al. (2014) Collapsin response mediator protein 5 

(CRMP5) induces mitophagy, thereby regulating mitochondrion 

numbers in dendrites. J. Biol. Chem. 289, 2261–2276. 

Martins, I. et al. (2014) Molecular mechanisms of ATP 

secretion during immunogenic cell death. Cell Death Differ. 

21, 79–91. 

Lu, B. et al. (2014) JAK/STAT1 signaling promotes HMGB1 

hyperacetylation and nuclear translocation. Proc. Natl. Acad. 

Sci. USA 111, 3068–3073. 

Li, Q. et al. (2014) Cited2, a transcriptional modulator 

protein, regulates metabolism in murine embryonic stem 

cells. J. Biol. Chem. 289, 251–263. 

Papa, L. et al. (2014) SirT3 regulates the mitochondrial 

unfolded protein response. Mol Cell Biol. 34, 699–710. 

Hong, S.W. et al. (2013) SVCT-2 in breast cancer acts 

as an indicator for L-ascorbate treatment. Oncogene 32, 

1508–1517. 

Zhai, H. et al. (2013) Inhibition of autophagy and tumor 

growth in colon cancer by miR-502. Oncogene 32, 

1570–1579. 

Brot, S. et al. (2014) Collapsin response mediator protein 5 

(CRMP5) induces mitophagy, thereby regulating mitochondrion 

numbers in dendrites. J. Biol. Chem. 289, 2261–2276. 

Target M P S 

Phospho-SQSTM1/p62 

(Thr269/Ser272) • 

Phospho-SQSTM1/p62 

(Ser403) 

• 

TECPR1 • 

TMEM49/VMP1 • 

ULK1 • • • 

Phospho-ULK1 (Ser317) • • 

Phospho-ULK1 (Ser467) • 

Phospho-ULK1 (Ser555) • 



UVRAG • • 

WIPI1 

• • 

WIPI2 

• 

Phospho-WIPI2 (Ser413) • 

Mealer, R.G. et al. (2014) Rhes, a striatal-selective protein 

implicated in Huntington disease, binds beclin-1 and activates 

autophagy. J. Biol. Chem. 289, 3547–3554. 

Huck, B. et al. (2014) Elevated protein kinase D3 (PKD3) 

expression supports proliferation of triple-negative breast 

cancer cells and contributes to mTORC1-S6K1 pathway 

activation. J. Biol. Chem. 289, 3138–3147. 

Conacci-Sorrell, M. et al. (2014) Stress-induced cleavage of 

Myc promotes cancer cell survival. Genes Dev. 28, 689–707. 

Huck, B. et al. (2014) Elevated protein kinase D3 (PKD3) 

expression supports proliferation of triple-negative breast 

cancer cells and contributes to mTORC1-S6K1 pathway 

activation. J. Biol. Chem. 289, 3138–3147. 

Talaber, G. et al. (2014) HRES-1/Rab4 promotes the formation 

of LC3(+) autophagosomes and the accumulation of 

mitochondria during autophagy. PLoS One 9, 84392. 

Lin, G. et al. (2014) Reduced Warburg effect in cancer cells 

undergoing autophagy: steady- state 1H-MRS and real-time 

hyperpolarized 13C-MRS studies. PLoS One 9, 92645. 

Nemati, F. et al. (2014) Targeting Bcl-2/Bcl-XL induces antitumor 

activity in uveal melanoma patient-derived xenografts. 


Ren, X.S. et al. (2014) Activation of the PI3K/mTOR pathway 

is involved in cystic proliferation of cholangiocytes of the PCK 

rat. PLoS One 9, e87660. 

Shiroto, T. et al. (2014) Caveolin-1 is a critical determinant 

of autophagy, metabolic switching, and oxidative stress in 

vascular endothelium. PLoS One, e87871. 

Uetake, R. et al. (2014) Adrenomedullin-RAMP2 system suppresses 

ER stress-induced tubule cell death and is involved 

in kidney protection. PLoS One 9, e87667. 

Chang, P.C. et al. (2014) Autophagy pathway is required for 

IL-6 induced neuroendocrine differentiation and chemoresistance 

of prostate cancer LNCaP cells. PLoS One 9, e88556. 

Lin, L. et al. (2014) Mechanical stress triggers cardiomyocyte 

autophagy through angiotensin II type 1 receptormediated 

p38MAP kinase independently of angiotensin II. 


Gu, W. et al. (2014) Ambra1 is an essential regulator of 

autophagy and apoptosis in SW620 cells: pro-survival role of 

Ambra1. PLoS One 9, e90151. 

Autophagy Signaling 

AMP: 

ATP 

AMPK 

Apoptosis 

Phagophore 

Atg16L1 

Amino 

Acids 

Atg16L1 

Macroautophagy 

PI3K-I/Akt 

Signaling 

GβL 

MAPK/Erk1/2 

Signaling 

Atg13 FIP200 

ULK1 

p150 

PI3K 

Class III 

Beclin-1 

Bcl-2 

Atg14 

Atg5 

Rubicon 

Atg12 

Atg12 

mTOR 

Ambra1 

Atg5 

Atg10 

Atg7 

p53/Genotoxic 

Stress 

Raptor 

PRAS40 

ub 

Cytoplasmic 

Contents 

Oxidative 

Stress 

Membrane 

Nucleation 

ub 

Mitophagy 

Mitochondrial 

Damage 

PARL 

BNIP3 

SQSTM1/p62 

BNIP3L/NIX 

+ NBR1 + 

ALFY 

Atg3 

Atg7 

Atg4 

Sequestration 

LC3-II 

LC3-I 

LC3 

PE 

PINK 

SQSTM1/p62 

NBR1 

Ambra1 


ub 

Parkin 

+ LC3-II 

Autophagosome 

Lysosome 

ub -targets 

Fusion 

Autophagolysosome 

Macroautophagy, often referred to as autophagy, is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic contents, abnormal 

protein aggregates, and excess or damaged organelles. Autophagy is generally activated by conditions of nutrient deprivation but has also been associated with physiological 

as well as pathological processes such as development, differentiation, neurodegenerative diseases, stress, infection, and cancer. The kinase mTOR is a critical regulator of 

autophagy induction, with activated mTOR (Akt and MAPK signaling) suppressing autophagy, and negative regulation of mTOR (AMPK and p53 signaling) promoting it. Three 

related serine/threonine kinases, UNC-51-like kinase -1, -2, and -3 (ULK1, ULK2, UKL3), which play a similar role as the yeast Atg1, act downstream of the mTOR complex. 

ULK1 and ULK2 form a large complex with the mammalian homolog of an autophagy-related (Atg) gene product (mAtg13) and the scaffold protein FIP200 (an ortholog of 

yeast Atg17). Class III PI3K complex, containing hVps34, Beclin-1 (a mammalian homolog of yeast Atg6), p150 (a mammalian homolog of yeast Vps15), and Atg14-like 

protein (Atg14L or Barkor) or ultraviolet irradiation resistance-associated gene (UVRAG), is required for the induction of autophagy. The Atg genes control autophagosome 

formation through Atg12-Atg5 and LC3-II (Atg8-II) complexes. Atg12 is conjugated to Atg5 in a ubiquitin-like reaction that requires Atg7 and Atg10 (E1 and E2-like enzymes, 

respectively). The Atg12-Atg5 conjugate then interacts noncovalently with Atg16 to form a large complex. LC3/Atg8 is cleaved at its C-terminus by Atg4 protease to generate 

the cytosolic LC3-I. LC3-I is conjugated to phosphatidylethanolamine (PE) also in a ubiquitin-like reaction that requires Atg7 and Atg3 (E1 and E2-like enzymes, respectively). 

The lipidated form of LC3, known as LC3-II, is attached to the autophagosome membrane. Autophagy and apoptosis are connected both positively and negatively, and extensive 

crosstalk exists between the two processes. During nutrient deficiency, autophagy functions as a pro-survival mechanism; however, excessive autophagy may lead to 

cell death, a process morphologically distinct from apoptosis. Several pro-apoptotic signals, such as TNF, TRAIL, and FADD, also induce autophagy. Additionally, Bcl-2 inhibits 

Beclin-1-dependent autophagy, thereby functioning both as a pro-survival and as an anti-autophagic regulator. 

Mitophagy is a selective autophagic process specifically designed for the removal of damaged or unneeded mitochondria from a cell. Upon mitochondrial damage, the protein 

PINK, which is continually degraded in the healthy state through the action of PARL, is stabilized and recruits the E3 ligase Parkin to initiate mitophagy. Polyubiquitination of 

mitochondrial membrane proteins by Parkin results in the recruitment of autophagy adaptor proteins SQSTM1/p62, NBR1, and Ambra1 that bind to LC3 via their LC3- 

interacting region (LIR). In addition, BNIP3 and BNIP3L/NIX, which also contain LIRs, directly recruit autophagic machinery by a ubiquitin-independent mechanism to induce 

autophagosome formation in certain cell types. 


Alers, S., Löffler, A.S., Wesselborg, S., and Stork, B. (2012) Mol. Cell. Biol. 32, 2–11. • Codogno, P., Mehrpour, M., and Proikas-Cezanne, T. (2012) Nat. Rev. Mol. Cell Biol. 

13, 7–12. • Ding, W.X. and Yin, X.M. (2012) Biol. Chem. 393, 547–564. • Feng, D., Liu, L., Zhu, Y., and Chen, Q. (2013) Exp. Cell. Res. 319, 1697–1705. • Jin, M. 

and Klionsky, D.J. (2014) FEBS Lett. 588, 2457–2463. • Papinski, D. and Kraft, C. (2014) Autophagy 10, 1338–1340. • Schneider, J.L. and Cuervo, A.M. (2014) Curr. 

Opin. Genet. Dev. 26, 16–23. 

© 2003–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Bingren Hu, University of Maryland School of Medicine, Baltimore, MD, for reviewing this diagram. 



97



Cell Cycle, Checkpoint 

Control, and DNA Damage 

Control of eukaryotic cell growth and division involves molecular circuits known as “checkpoints” that 

ensure proper timing of cellular events. Passage through a checkpoint from one cell cycle phase to the 

next requires a coordinated set of proteins that monitor cell growth and DNA integrity. Uncontrolled cell 

division or propagation of damaged DNA can contribute to genomic instability and tumorigenesis. 

Phases of the Cell Cycle 

G2/M Checkpoint 

The G2/M checkpoint prevents cells containing damaged DNA from entering mitosis (M). Activated 

CDK1 (cdc2) bound to cyclin B promotes entry into M-phase. Wee1 and Myt1 kinases and cdc25 

phosphatase competitively regulate CDK1 activity; Wee1 and Myt1 inhibit CDK1 and prevent entry into 

M-phase, while cdc25 removes inhibitory phosphates. DNA damage activates multiple kinases such 

as ATM/ATR, DNA-PK, HIPK2 that phosphorylate kinases Chk1/2 and tumor suppressor protein p53. 

Chk1/2 kinases stimulate Wee1 activity and inhibit cdc25C, preventing entry into M-phase. Phosphorylation 

of p53 promotes dissociation between p53 and MDM2 and allows binding of the transcription 

factor to DNA. 

UV treatment results 

in phosphorylation of 

ATM at Ser1981. 

kDa 

200 

Phospho-ATM 

(Ser1981) 

G2 

Preparation 

for Mitosis 

Prophase 

Metaphase 

Anaphase 

M 

Cyclin B1/CDK1 complex is expressed in replicating Jurkat cells. 

Cyclin B1 (D5C10) XP ® Rabbit mAb #12231: Flow cytometric analysis of 

Jurkat cells using #12231 and Propidium Iodide (PI)/RNase Staining Solution 

#4087 (DNA content). Anti-rabbit IgG (H+L), F(ab’) 2 

Fragment (Alexa Fluor ® 

488 Conjugate) #4412 was used as a secondary antibody. 

Cyclin B1 

10 4 

10 3 

10 2 

140 

100 

80 

200 

ATM 

Telophase 

10 1 

140 

G 0 

10 0 0 200 400 600 800 1000 

DNA (PI) 

100 

S 

DNA 

Replication 

Preparation 

for DNA 

Synthesis 

G1 

UV treatment results in nuclear translocation of Phospho-p53. 

Phospho-p53 (Ser15) (16G8) Mouse 

mAb (Alexa Fluor ® 555 Conjugate) 

#9481: Confocal IF analysis of HT-29 

cells, untreated (left) or UV-treated 

(right), using #9481 (red). Actin filaments 

were labeled with Alexa Fluor ® 

488 Phalloidin #8878 (green). 

80 

– + UV 

Phospho-ATM (Ser1981) (D6H9) 


extracts from 293 cells, untreated or 

UV-treated (100 mJ, 4 hr recovery), 

using #5883 (upper) or ATM (D2E2) 


CDK inhibitor p27 

Kip1 is expressed 

in quiescent cells 

and degraded in 

mitotic cells. 

p27 Kip1 (D69C12) XP ® Rabbit 

mAb #3686: Flow cytometric 

analysis of Jurkat cells using 

#3686 versus Propidium Iodide 

(PI)/RNase Staining Solution 

#4087. Anti-rabbit IgG (H+L), 

F(ab’) 2 Fragment (Alexa Fluor ® 

488 Conjugate) #4412 was 

used as a secondary antibody. 

G1/S Checkpoint 

The G1/S checkpoint controls progression of cells through the restriction point (R) into the DNA 

synthesis S-phase. During G1, the tumor suppressor Rb binds and inhibits transcription factor E2F. 

Phosphorylation of Rb by cyclin-bound cyclin dependent kinases (CDK) in late G1 induces dissociation 

of Rb and permits E2F-mediated transcription of S-phase-promoting genes. Responding to upstream 

signals, INK4 and Kip/Cip family inhibitors control CDK activity and prevent entry into S-phase. DNA 

damage activates response pathways through ATM/ATR and Chk1/2 kinases to block CDK activity, 

leading to cell cycle arrest and DNA repair or cell death. 

p27 Kip1 

10 4 

10 3 

10 2 

10 1 

10 0 0 200 400 600 800 1000 

DNA (PI) 

Serum treatment results in 

phosphorylation of Rb at Ser807/811. 

Phospho-Rb (Ser807/811) 

(D20B12) XP ® Rabbit mAb 

#8516: WB analysis of extracts 

from WI-38 cells using #8516. 

Lanes 

1. Serum-starved for 3 days (-) 

2. Serum-starved for 3 days 

followed by 10% serum for 

2 days (+) 

kDa 

200 

140 

100 

80 

60 

1 2 

Phospho-Rb 

(Ser807/811) 

Spindle Checkpoint 

As mitosis proceeds, mitotic spindles are assembled that connect the kinetochore regions of sister 

chromatids to the microtubules within the spindle, centering the chromatids along the metaphase 

plate. Many proteins comprise the kinetochore and play a role in this process, including the histone H3 

variant CENP-A, survivin, and Aurora B kinase, which is itself regulated by the microtubule nucleating 

protein TPX2. The spindle checkpoint ensures proper chromatid attachment prior to progression from 

metaphase to anaphase. The SCF and APC/C protein complexes play prominent roles in the spindle 

checkpoint, with APC-cdc20 initiating the entry into anaphase by promoting ubiquitin-mediated degradation 

of multiple substrates, including cyclin B and the regulatory protein securin. 

Chemical Modulators for Studies of the Cell Cycle and DNA Damage 

#9886 Docetaxel Inhibits microtubule depolymerization; prevents cell division 

#5927 Doxorubicin Inhibits DNA and RNA synthesis by intercalating the DNA helix; inhibits topoisomerase I 

#2200 Etoposide Inhibits topoisomerase II resulting in DNA breakage; induces apoptosis 

#2194 MG-132 Potent inhibitor of the proteasome and calpain 

#2190 Nocodazole Inhibits microtubule polymerization; induces cell cycle arrest in G2/M-phase 

#9807 Paclitaxel Inhibits microtubule depolymerization; prevents cell division 

#9885 Roscovitine Potent and selective inhibitor of CDK1, 2, and 5 (ATP-competitive) 

TPX2 regulates 

activity of Aurora B, 

a kinase critical for 

proper chromatid 

attachment to the 

mitotic spindle. 

TPX2 (D2R5C) XP ® Rabbit mAb 

#12245: Confocal IF analysis of MCF7 

cells using #12245 (green). Actin filaments 

were labeled with DY-554 phalloidin 

(red). Blue pseudocolor = DRAQ5 ® 



www.cellsignal.com/cstcellcycle 99



nodes within cell cycle signaling 


in cell cycle research. Primary 











Aurora Antibody Sampler Kit #3875 contains antibodies to several 

targets important for investigating the G2/M phase of the cell cycle. 

Phospho-Aurora A (Thr288) (C39D8) 

Rabbit mAb #3079: Confocal IF 

analysis of mitotic HeLa cells during 

metaphase (left) and anaphase (right) 

using #3079 (red) and Phospho-Histone 

H3 (Ser10) (6G3) Mouse mAb #9706 

(green). Blue pseudocolor = DRAQ5 ® 


#3079 is a component of the 

Aurora Antibody Sampler Kit #3875. 


Bertoli, C., Skotheim, J.M., and de Bruin, R.A. (2013) Nat. Rev. Mol. Cell Biol. 14, 518–528. • Bretones, G., Delgado, M.D., 

and León, J. (2014) Biochim. Biophys. Acta. April 1 [Epub ahead of print] • Choi, Y.J. and Anders, L. (2014) Oncogene 

33, 1890–1903. • Duronio, R.J. and Xiong, Y. (2013) Cold Spring Harb. Perspect. Biol. 5, a008904. • Johnson, A. and 

Skotheim, J.M. (2013) Curr. Opin. Cell Biol. 25, 717–723. • Lord, C.J. and Ashworth, A. (2012) Nature 481, 287–294. • 

Maréchal, A. and Zou, L. (2013) Cold Spring Harb. Perspect. Biol. 5, a012716. • Yasutis, K.M. and Kozminski, K.G. (2013) Cell 

Cycle 12, 1501–1509. 

Commonly Studied Cell Cycle Targets 


APC6 

• 

Ape1 

• 

Artemis 

• 

ATM • • 

Phospho-ATM (Ser1981) • 

ATR • • • 

Phospho-ATR (Ser428) • 

ATRIP 

• 

Phospho-ATRIP (Ser224) • 

Aurora A • • • 

Phospho-Aurora A (Thr288) • • 

• • 

Phospho-Aurora A (Thr288)/Aurora B 

(Thr232)/Aurora C (Thr198) 

Aurora B • • 

BACH1/BRIP1 

• 

Phospho-BACH1/BRIP1 (Thr1133) • 

BLM 

• 

Bora 

• 

BRCA1 • • • 

Phospho-BRCA1 (Ser1524) • 

BrdU 

• 

BRE 

• 

Bub1 

• 

Bub1B 

• • 

Bub3 

• • 

Cdc2 • • 

Phospho-Cdc2 (Thr14) • 

Phospho-Cdc2 (Tyr15) • • • 

Phospho-Cdc2 (Thr161) • 

CDC20 

• 

Phospho-CDC20 (Ser51) • 

cdc25A 

• 

cdc25B 

• 

cdc25C 

• 

Phospho-cdc25C (Thr48) • 

Phospho-cdc25C (Ser198) • 


Phospho-cdc25C (Ser216) • • 

Cdc6 

• 

Cdc7 

• 

CDC37 

• 

Cdc45 

• • 

CDC73 

• • 

CDK2 • • 


CDK4 

• 

CDK6 

• 

CDK7 

• • 

CDK8 • • 

CDK9 • • 


PITSLRE/CDK11 

• 

CDK12 

• 

CDT1 

• • 

CENP-T 

• 

CHFR 

• • 

Chk1 • • • 

Phospho-Chk1 (Ser280) • 


Phospho-Chk1 (Ser317) • • • 

Phospho-Chk1 (Ser345) • • • 

Chk2 • • • 


Phospho-Chk2 (Ser33/35) • 

Phospho-Chk2 (Thr68) • • • • 


CK2α • • 

Claspin 

• 

Phospho-CLASP2 (Ser1234) • 

CP110 

• 

CtIP 

• 

CUEDC2 

• 



Cyclin A2 

• 

Cyclin B1 • • • 

Phospho-Cyclin B1 (Ser116) • 



Cyclin D1 • • • 

Phospho-Cyclin D1 (Thr286) • • 

Cyclin D2 

• • 

Cyclin D3 

• 

Cyclin E1 

• 

Phospho-Cyclin E1 (Thr62) • 

Cyclin E2 

• 

Cyclin H 

• 

DNA-PK 

• • 

Phospho-DNA-PK (Ser2056) • 

DNA Polymerase γ • 

DNA Polymerase η • 

DYRK2 

• • 

E2F-1 

• 

EAPP 

• 

Eg5 

• • 

ENSA 

• • 

• 

Phospho-ENSA (Ser67)/ARPP19 

(Ser62) 

ERCC1 

• • 

FEN-1 

• 

FOXM1 

• 

Phospho-FOXM1 (Ser35) • 

GADD45 α 

• 

Geminin 

• 

HR6A/HR6B 

• 

INCENP 

• 

p18 INK4C 

• 

Ki-67 • • 

Ku70 

• • 

Ku80 

• • 

LATS1 

• • 

Phospho-LATS1 (Ser909) • 

Phospho-LATS1 (Thr1079) • 

LATS2 

• • 

MAD2L1 

• 

MASTL 

• 

MCM2 

• • 

Phospho-MCM2 (Ser139) • • 

Phospho-MDM2 (Ser166) • 

MCM3 

• • 

Phospho-MCM3 (Ser112) • 

MCM4 

• • 

MCM7 

• • 

MERIT40 

• • 

Phospho-MERIT40 (Ser29) • 

Microcephalin-1/BRIT1 • 

MGMT 

• 

MLH1 

• 

Mre11 

• • 

Phospho-Mre11 (Ser676) • 

MSH2 

• 

MSH6 

• • 

MYH 

• • 

Myt1 

• 


Phospho-Myt1 (Ser83) • 

NCAPD3 

• 

NEK7 

• 

NIPA 

• 

NPM 

• 

Phospho-NPM (Ser4) • 

Phospho-NPM (Thr95) 

• 

Phospho-NPM (Thr199) • 

NuMA 

• • 

Phospho-NuMA (Ser395) • 

ORC1 

• 

ORC2 

• 

ORC6 

• 

p14 ARF 

• 

p21 Waf1/Cip1 • • • • 

p27 Kip1 

• • • • • 

p48 Primase 

• 

p53 

• • • • • 

Acetyl-p53 

• 

Acetyl-p53 (Lys379) 

• 

Acetyl-p53 (Lys382) 

• 

Phospho-p53 (Ser6) 

• 


• 

Phospho-p53 (Ser15) • • • • 

Phospho-p53 (Thr18) 

• 


• 


• 


• 


• 

Phospho-p53 (Thr81) 

• 

Phospho-p53 (Ser315) • 

Phospho-p53 (Ser392) • 

53BP1 

• 

Phospho-53BP1 (Ser25/29) • 

Phospho-53BP1 (Thr543) • 

Phospho-53BP1 (Ser1618) • 

Phospho-53BP1 (Ser1778) • 

p57 Kip2 

• 

Phospho-p57 Kip2 (Thr310) • 

p58 Primase 

• 

p63-α 

• • 

Phospho-p63 (Ser160/162) • 

p73 

• 

Phospho-p73 (Tyr99) 

• 

p95/NBS1 

• 

Phospho-p95/NBS1 (Ser343) • 

PBK/TOPK 

• 

Phospho-PBK/TOPK (Thr9) • 

PCNA • • 

Ubiquityl-PCNA (Lys164) • 

PCTAIRE 1 

• 

PELO 

• 

PHB1 

• 

PICH 

• 

Pin1 

• 

PLK1 • • • 

Phospho-PLK (Ser137) • 

Phospho-PLK (Thr210) • • 

PLK3 

• • 

Phospho-PNK1 (Ser114/Thr118)) • 


152 


CST antibodies for phospho-p53 





Godinho, S.A. et al. (2014) 

Oncogene-like induction of cellular 

invasion from centrosome amplification. 

Nature 510, 167–171. 

Lee, T.H. et al. (2014) Coordinated 

regulation of XPA stability by ATR and 

HERC2 during nucleotide excision 

repair. Oncogene 33, 19–25. 

Gad, H. et al. (2014) MTH1 inhibition 

eradicates cancer by preventing 

sanitation of the dNTP pool. Nature 

508, 215–221. 

Rashi-Elkeles, S. et al. (2014) Parallel 

profiling of the transcriptome, cistrome, 

and epigenome in the cellular 

response to ionizing radiation. Sci. 

Signal. 7, rs3. 

Baresova, P. et al. (2014) p53 tumor 

suppressor protein stability and 

transcriptional activity are targeted 

by Kaposi’s sarcoma-associated 

herpesvirus-encoded viral interferon 

regulatory factor 3. Mol. Cell. Biol. 

34, 386–399. 

Cam, M. et al. (2014) p53/TAp63 

and AKT regulate mammalian target 

of rapamycin complex 1 (mTORC1) 

signaling through two independent 

parallel pathways in the presence of 

DNA damage. J. Biol. Chem. 289, 

4083–4094. 

Zheng, H. et al. (2014) p53 promotes 

repair of heterochromatin DNA by 

regulating JMJD2b and SUV39H1 

expression. Oncogene 33, 734–744. 

Shi, Y. et al. (2014) ROS-dependent 

activation of JNK converts p53 into an 

efficient inhibitor of oncogenes leading 

to robust apoptosis. Cell Death 

Differ. 21, 612–623. 

Penicud, K. et al. (2014) DMAP1 is an 

essential regulator of ATM activity and 

function. Oncogene 33, 525–531. 

Endo, F. et al. (2014) A compensatory 

role of NF-kappaB to p53 in response 

to 5-FU-based chemotherapy for 

gastric cancer cell lines. PLoS One 

9, e90155. 

Leikam, C. et al. (2014) Cystathionase 

mediates senescence evasion in 

melanocytes and melanoma cells. 

Oncogene 33, 771–782. 

www.cellsignal.com/cstcellcycle 101


UV treatment results 

in ATM/ATR-mediated 


Chk1, initiating the DNA 

damage checkpoint. 

A 

B 

C 

Phospho-Chk1 (Ser317) (D12H3) XP ® 

Rabbit mAb #12302: Confocal IF analysis 

of HeLa cells, untreated (A), UV-treated 

(B), or UV and λ phosphatase-treated (C), 

using #12302 (green). Actin filaments were 



PP1α 

• 

Phospho-PP1α (Thr320) • 

PP2A A subunit • • • 

PP2A B subunit 

• • 

PPP2R2A 

• 

PP2A C subunit 

• • 

Nonmethylated PP2A C Subunit • 

PPP1CB 

• 

PTPA/PPP2R4 

• • 

PPP2R5D 

• 

PP5 

• 

Rad17 

• 

Phospho-Rad17 (Ser635) • 

Phospho-Rad17(Ser645) • 

Rad18 

• 

Phospho-Rad18 (Ser403) • 

RAD21 

• 

Rad50 

• 

Rad51 

• 

Rb • • • 

Phospho-Rb (Ser608) • • 

Phospho-Rb (Ser780) • • • 

Phospho-Rb (Ser795) 

• 

Phospho-Rb (Ser807/Ser811) • • • 

Rb-like 1 

• 

Phospho-RCC1 (Ser11) • 

RecQL1 

• 

RecQ4 

• 

RecQL5 

• 

RPA70/RPA1 

• • 

RPA32/RPA2 

• 

Rpb1 CTD 

• 

Phospho-Rpb1 CTD (Ser2/5) • 

RRM1 

• • 

Securin 

• 

Sestrin-2 

• 

SLBP 

• 

Cell Cycle/Checkpoint Control Kinase-Disease Associations 


ATM Atypical Cancer 

CNS 

LOF Mut LOF mutations associated with ataxia talangiectasia [OMIM:208900], causing 

progressive loss of motor control (ataxia), dilation of superficial blood vessels 

(telangiectasia), cancer and immune deficiency. Approximately 30% of 

cases develop tumors, mostly lymphomas and leukemias, due to defects in 

DNA damage repair. Somatic mutations seen in leukemias and lymphomas. 

OMIM:607585. 

ATR Atypical Cancer 

Development 

Virology 

Mut, Splice 

Functions in DNA damage responses. A splice-altering mutation seen in 

cases of Seckel syndrome [OMIM:210600], featuring dwarfism and mental 

retardation. Heterozygous knockout mice are cancer-prone. Mutations seen 

in cancers of the stomach (Medline:11691784) and endometrium (Medline:12124347), 

tumors with high mutation rates due to microsatellite instability. 

May also be required for retroviral DNA integration (Medline:12679521). 

OMIM:601215. 

AurB Other Cancer OE Required for chromosome segregation and cytokinesis. Overexpressed in 

colorectal and other cancer cell lines (Medline:9809983) and thought to cause 

aneuploidy via histone phosphorylation (Medline:12884918). OMIM:604970. 

CDC2 

(CDK1) 


SMC1 

• • 

Phospho-SMC1 (Ser360) • 

Phospho-SMC1 (Ser957) • • 

SMC2 

• 

SMC3 

• 

SMC4 

• 

SMG-1 

• • 

Spartin 

• 

STAG2 

• • 

TACC3 

• 

Phospho-TACC3 (Ser558) • • 

TCTP 

• 

TERF2IP 

• 

TIF1β 

• • 

Phospho-TIF1β (Ser824) • 

TLK1 

• 

Phospho-TLK1 (Ser743) • 

TPX2 

• • 

Trf1 

• 

Trf2 

• • 

TTK 

• • 

VCP 

• 

VRK1 

• 

VRK3 

• 

Wee1 

• • 

Phospho-Wee1 (Ser642) • 

WIP1 

• 

WRN 

• 

XLF 

• 

XPB 

• 

XPC 

• 

XPD 

• 

XPF 

• 

XRCC1 

• 

CMGC Cancer Act, Splice Cell cycle checkpoint. Activated in many cancers including colon, liver and 

breast (Medline:10091728, 12100577, 11091571). The delta T isoform, 

which lacks a regulatory region, is expressed in breast cancer. Inhibition in 

cancer cells may drive cells into apoptosis (Medline:12150824). May also 

drive cell migration (Medline:12771130). Inhibitors: BMS-265246, BMS- 

265246-01 (Bristol-Myers Squibb). OMIM:116940. 



CDK2 CMGC Cancer Cell cycle checkpoint and part of the Rb pathway disregulated in most tumors 

(Medline:12888290). Target of several candidate cancer drugs. However, 

inhibition does not always prevent cancer cell growth (Medline:12676582) 

due possibly to CDK redundancy. Inhibitors: BMS-265246, BMS-265246-01 

(Bristol-Myers Squibb), R-roscovitine (CYC200, CYC202) (Cyclacel; Ph. 2), 

SU9516 (Sugen), R547 (Roche), L868276. OMIM:116953. 

CDK4 CMGC Cancer Act, GOF 

Mut, Amp, 

Meth 

Point mutations found in somatic and familial melanoma. Amplified in 

sarcomas (Medline:9703873, 9935200), glioma (Medline:14756442) and 

lymphoma (Medline:12203778). Amplified, methylated or deleted in head 

and neck squamous cell carcinoma (Medline:14586645). Overexpression 

drives epithelial tumors in mice (Medline:14647432). Disruption makes 

mice resistant to cancer (Medline:12435633). Inhibitor: PD332991 (Onyx). 

OMIM:123829. 

CDK5 CMGC Neurodegeneration SNP Implicated in the pathology of neurofibrillary tangles and formation of senile 

plaques, hallmarks of Alzheimer disease (AD). Induces tau phosphorylation 

and aggregation and neurofibrillary tangle deposition and neurodegeneration 

in in vitro and in vivo animal models. Brain samples from AD patients show 

elevated CDK5 activity, possibly induced by Aβ amyloid. SNPs show weak AD 

association (Medline:18350355) OMIM:123831. 

CDK6 CMGC Cancer OE, Trans Overexpressed and/or disrupted by translocation in leukemias, lymphomas 

and other cancers; amplified in gliomas (Medline:9102208) and rodent 

cancers (Medline:12538879, 11719459). OMIM:603368. 

CDK9 CMGC Cardiovascular 


CDKL5 CMGC CNS 

Development 

CHK1 

(CHEK1) 

Expr 

Mut, Trans 

Transcriptional elongation factor and cofactor for HIV Tat protein; RNAi against 

CDK9 blocks HIV replication and inhibitors also block varicella zoster replication. 

Mediates signals leading to cardiac hypertrophy (Medline:12695656). 

Inhibitor: Flavopiridol. (Aventis) OMIM:603251. 

Missense, splice and truncating mutations linked to the neurodevelopmental 

Rett Syndrome [OMIM:312750]. A chromosomal translocation which silences 

the gene is associated with severe X-linked infantile spasm syndrome 

[OMIM:308350]. OMIM:300203. 

CAMK Cancer Mut Cell cycle G2 checkpoint kinase implicated in resistance to apoptosis in 

response to chemotherapy. Inhibitors under development to chemosensitize 

tumors. Somatic mutations found in stomach tumors (Medline:11691784) 

and in colon and endometrial tumors, where CHK1 may be a target of 

microsatellite instability (Medline:14657665). Inhibitors: SB218078, UNC-01. 

OMIM:603078. 

CHK2 CAMK Cancer Mut Tumor suppressor involved in DNA damage and cell cycle arrest. LOF mutants 

cause Li-Fraumeni syndrome [OMIM:151623], a highly penetrant familial 

cancer phenotype also caused by p53 mutations. Familial mutations also associated 

with prostate and breast cancer, and mutations also seen in a variety 

of sporadic cancers and cell lines. OMIM:604373. 

CK1δ 

CK1α 

CK1ε 

(CSNK1ε) 

CK1 Neurodegeneration Several CK1 isoforms including CK1δ and CK1α associate with and 

phosphorylate tau, which forms the neurofibrillary tangles of Alzheimer’s 

disease. Tau phosphorylation and microtubule association is inhibited by 

the CK1 inhibitor IC261 (Medline:14761950). CK1 phosphorylation is 

involved in trafficking of the Alzheimer’s plaque component, β secretase 

(Medline:11278841). CK1 levels are increased in Alzheimer’s brains. CK1δ is 

also associated with tau tangles in several other neurodegenerative diseases 

(Medline:10924763). CK1δ mRNA and protein are upregulated in Alzheimer’s 

brain regions with most pathology (Medline:10814741). CK1 also phosphorylates 

the Parkinson’s-associated α synuclein protein (Medline:10617630). 

OMIM:600864, 60050. 

CK1 

CK2α1 Other 

CK2α2 

(CSNK2α1/2) 

Behavior 

Cancer 

Cancer 

Circadian Rhythm 


SNP, Mut, 

LOH 

OE, Act 

Mutations in hamster and Drosophila orthologs have circadian rhythm phenotypes 

as the circadian gene period (per) is a substrate in both human and fly. 

A coding SNP variant in human increases CK1e activity and is negatively associated 

with circadian disorder (Medline:15187983). LOF mutations and LOH 

seen in mammary ductal carcinoma (Medline:14871824). OMIM:600863. 

Two gene products (α1/α>2 or α/α) complex with each other and a regulatory 

β subunit. The isoforms are rarely distinguished from each other in 

publications. Transgene expression in mice leads to lymphoma and activation 

by bovine parasites leads to fatal lymphoproliferation (Medline: 7846532). 

Expression and activity are elevated in lung tumors (Medline:15355908, 

12017291) and breast tumors (Medline:11423974). Mouse transgene 

causes mammary gland hyperplasia. Antisense drives apoptosis of tumor 

cell lines (Medline:11827168) and xenografts (Medline:14965269). 

Involved in DNA break repair by phosphorylation of scaffold protein XRCC1 

(Medline:15066279), phosphorylation of BRCA1 (Medline:10403822) and 

phosphorylation of p53 in response to UV irradiation. The Drosophila CK2 

ortholog (Timekeeper) is involved in circadian regulation. Phosphorylates and 

binds to a major component of the inclusion bodies seen in Parkinson patients 

(Medline:14645218). Inhibitors: DMAT, Antisense, P15 peptide, 4,5,6,7-tetrabromobenzotriazole 

(TBB). OMIM:115440, 115442. 

Molecular: Act Activated • Amp Amplified • Del Deleted • Expr Expression • GOF Gain-of-function • Inh Inhibitor Studies • LOF Loss-offunction 

• LOH Loss-of-heterozygosity • Meth Methylation • Model model organism studies • Mut Mutation OE Overexpression • SNP Single 

Nucleotide Polymorphism • Splice Splicing change • Trans Translocation 


Cancer 

Research 

Please visit our website to learn 

more about the scientific tools and 

educational resources we have online 

for cancer signaling and proteomic 

analysis, including discussion of key 

disease drivers. 

www.cellsignal.com/cancerguide 



Cell Cycle Control: G1/S Checkpoint 

Ultra Violet 

Stress Response 

Growth 

Factor 

Withdrawal 


Hormones 

p19 INK4D 

GSK-3β 

SCF 


p18 INK4C 

Myc 

G1-PHASE 

Replicative 

Senescence 

p16 INK4A 

Cyclin D 

BMI1 

CDK4/6 

TGF-β 

p15 INK4B 

Myc 

Smad3 

Smad4 

cdc25A 

Growth Factor 

Receptor Activation 

R 

Akt 

FoxO1/3 

p27 Kip1 

CDK2 

Cyclin E 

Myc 

S-PHASE 

p21 Cip1 

Myc 

p53 

ATM/ 

ATR 

Replicative 

Senescence 

SCF 

DNA 

Damage 

Chk1/2 



Cell Cycle Control: G2/M DNA Damage Checkpoint 

HIPK2 

Nuclear Export, 


Nucleolar 

Sequestration 

or 

p53 Stabilization 

p19 Arf cdc25A 

IR UV 

DNA Repair 

DNA-PK 

ATM/ 

ATR 

Caffeine 

WIP1 

MDM4 

MDM2 p53 

Chk2 

TopoII BRCA1 14-3-3σ Reprimo GADD45 p21 Cip1 CDK7 

Nuclear 

Exclusion 

TRIP12 

MDM2 

p53 

p300/ 

PCAF 

WIP1 

BRCA1 

Chk1 

cdc25 


Critically Short 

Telomeres 

POT1 TRF2 

PLK1 

Myt1 

Rad50 

NBS1 

Mre11 

BRCA1 

c-Abl 

14-3-3 

cdc25A/C 

cdc25A/B 

(MRN) 

SCF 

SCF 

FANCD2 

Rad51 

p90RSK 

DNA 

Repair 

Rad52 

Nuclear Exclusion 


AurA 

Bora 

FoxO1 

DBE 

Bim 

FasL 

TRAIL 

Apoptosis 

Suv39H1 

Abl 

Rb HDAC 

E2F 

DP-1 

OFF 

Rb 

E2F 

DP-1 

ON 

E2F/DP Target Genes: 

Cyclin E/A, E2F-1/2/3, 

cdc2, c-Myc, p107, 

RanGAP, TK, DHFR, 

PCNA, H2A, etc. 

G2-PHASE 

cdc2 

Cyclin B 

Wee1 

M-PHASE 


The primary G1/S cell cycle checkpoint controls the commitment of eukaryotic cells to transition through the G1 phase to enter into the DNA synthesis S phase. Two cell 

cycle kinase complexes, CDK4/6-Cyclin D and CDK2-Cyclin E, work in concert to relieve inhibition of a dynamic transcription complex that contains the retinoblastoma protein 

(Rb) and E2F. In G1-phase uncommitted cells, hypo-phosphorylated Rb binds to the E2F-DP1 transcription factors forming an inhibitory complex with HDAC to repress key 

downstream transcription events. Commitment to enter S-phase occurs through sequential phosphorylation of Rb by Cyclin D-CDK4/6 and Cyclin E-CDK2 that dissociates 

the HDAC-repressor complex, permitting transcription of genes required for DNA replication. In the presence of growth factors, Akt can phosphorylate FoxO1/3, which inhibits 

their function by nuclear export, thereby allowing cell survival and proliferation. Importantly, a multitude of different stimuli exert checkpoint control, including TGF-β, DNA 

damage, replicative senescence, and growth factor withdrawal. These stimuli act though transcription factors to induce specific members of the INK4 or Kip/Cip families of 

cyclin dependent kinase inhibitors (CKIs). Notably, the oncogenic polycomb protein Bmi1 acts as a negative regulator of INK4A/B expression in stem cells and human cancer. 

In addition to regulating CKIs, TGF-β also inhibits cdc25A transcription, a phosphatase directly required for CDK activation. At a critical convergence point with the DNAdamage 

checkpoint, cdc25A is ubiquitinated and targeted for degradation via the SCF ubiquitin ligase complex downstream of the ATM/ATR/Chk-pathway. However, timely 

degradation of cdc25A in mitosis (M-phase) via the APC ubiquitin ligase complex allows progression through mitosis. Furthermore, growth factor withdrawal activates GSK-3β 

to phosphorylate Cyclin D, which leads to its rapid ubiquitination and proteasomal degradation. Collectively, ubiquitin/proteasome-dependent degradation and nuclear export 

are mechanisms commonly used to effectively reduce the concentration of cell cycle control proteins. Importantly, Cyclin D1/CKD4/6 complexes are explored as therapeutic 

targets for cancer treatment as researchers have found this checkpoint to be invariantly deregulated in human tumors. 


Besson, A., Dowdy, S.F., and Roberts, J.M. (2008) Dev. Cell. 14, 159–169. • Gil, J. and Peters, G. (2006) Nat. Rev. Mol. Cell Biol. 7, 667–677. • Malumbres, M. and 

Barbacid, M. (2009) Nat. Rev. Cancer 9, 153–166. • Musgrove, E.A., Caldon, C.E., Barraclough, J., Stone, A., and Sutherland, R.L. (2011) Nat. Rev. Cancer 11, 558–572. 

• Skaar, J.R. and Pagano, M. (2009) Curr. Opin. Cell Biol. 21, 816–824. • Sparmann, A. and van Lohuizen, M. (2006) Nat. Rev. Cancer 6, 846–856. • Tzivion, G., 

Dobson, M., and Ramakrishnan, G. (2011) Biochim. Biophys. Acta. 1813, 1938–1945. • van den Heuvel, S. and Dyson, N.J. (2008) Nat. Rev. Mol. Cell Biol. 9, 713–724. 

• Yang, J.Y. and Hung, M.C. (2009) Clin. Cancer Res. 15, 752–757. 

The G2/M DNA damage checkpoint serves to prevent the cell from entering mitosis (M-phase) with genomic DNA damage. Specifically, the activity of the Cyclin B-cdc2 (CDK1) 

complex is pivotal in regulating the G2-phase transition wherein cdc2 is maintained in an inactive state by the tyrosine kinases Wee1 and Myt1. It is thought that coordinated 

action of the kinase Aurora A and the cofactor Bora activate PLK1 as cells approach the M-phase, which in turn activates the phosphatase cdc25 and downstream cdc2 activity, 

hence establishing a feedback amplification loop that efficiently drives the cell into mitosis. Importantly, DNA damage cues activate the sensory DNA-PK/ATM/ATR kinases, which 

relay two parallel cascades that ultimately serve to inactivate the Cyclin B-cdc2 complex. The first cascade rapidly inhibits progression into mitosis: the Chk kinases phosphorylate 

and inactivate cdc25, which prevents activation of cdc2. The slower second parallel cascade involves phosphorylation of p53 and allows for its dissociation from MDM2 and 

MDM4 (MdmX), which activates DNA binding and transcriptional regulatory activity, respectively. The transcriptional ability of p53 is further augmented through acetylation by the 

co-activator complex p300/PCAF. The second cascade constitutes the p53 downstream-regulated genes including: 14-3-3, which binds to the phosphorylated Cyclin B-cdc2 

complex and exports it from the nucleus; GADD45, which binds to and dissociates the Cyclin B-cdc2 complex; and p21 Cip1, an inhibitor of a subset of the cyclin-dependent 

kinases including cdc2. Recent data suggest an important role for the p53-regulated WIP1 phosphatase that acts as a critical dampener of DNA damage signaling in cancer. 

In human cancer, researchers have found p53 to be commonly mutated, indicating that this checkpoint is a critical barrier to tumor formation. In addition, sporadic and familial 

mutations in the DNA-repair proteins such as the BRCA-family, ATM, and the Fanconi Anemia proteins further highlight this as a key tumor suppressor checkpoint. 


Abbas, T. and Dutta, A. (2009) Nat. Rev. Cancer 9, 400–414. • Al-Ejeh, F., Kumar, R., Wiegmans, A., Lakhani, S.R., Brown, M.P., and Khanna, K.K. (2010) Oncogene 29, 

6085–6098. • Boutros, R., Lobjois, V., and Ducommun, B. (2007) Nat. Rev. Cancer 7, 495–507. • Ciccia, A. and Elledge, S.J. (2010) Mol. Cell 40, 179–204. • Freed- 

Pastor, W.A. and Prives, C. (2012) Genes Dev. 26, 1268–1286. • Huen, M.S., Sy, S.M., and Chen, J. (2010) Nat. Rev. Mol. Cell Biol. 11, 138–148. • Junttila, M.R. and 

Evan, G.I. (2009) Nat. Rev. Cancer 9, 821–829. • Lens, S.M., Voest, E.E., and Medema, R.H. (2010) Nat. Rev. Cancer 10, 825–841. • Kee, Y. and D’Andrea, A.D. (2010) 

Genes Dev. 24,1680–1694. • Nam, E.A. and Cortez, D. (2011) Biochem. J. 436, 527–536. • Reinhardt, H.C. and Yaffe, M.B. (2009) Curr. Opin. Cell Biol. 21, 245–255. 

© 2002–2015 Cell Signaling Technology, Inc. • We would like to thank Dr. Hans Widlund, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, for reviewing this diagram. 





VE-cadherin is 

expressed in 

adherens junctions 

of endothelial cells. 

VE-Cadherin (D87F2) XP ® Rabbit mAb 

#2500: Confocal IF analysis of HUVE cells 

(top) and HeLa cells (bottom) using #2500. 

Actin filaments were labeled with DY-554 

phalloidin (red). Blue pseudocolor = 


Adhesion and Extracellular Matrix 

Cells can form a number of connections with the cells and matrix in their surrounding environment, 

including adherens junctions (cell-cell), tight junctions (impermeable cell-cell), and focal adhesions 

(cell-matrix). 

Adherens Junctions 

Adherens junctions are cell-cell connections mediated through a cadherin-catenin complex composed 

of cadherin, β-catenin, and α-catenin. The connection between cell junctions and the cytoskeleton is 

more dynamic than originally considered, relying on multiple, weak associations between the cadherincatenin 

complex and the actin cytoskeleton or on other membrane-associated proteins (i.e. nectin and 

afadin). Monomeric α-catenin binds β-catenin at adherens junctions, and upon release, forms α-catenin 

dimers that promote actin bundle formation. The transition from branched actin networks to bundled 

actin filaments correlates with the creation of mature, strong adherens junctions, and a decrease in 

membrane lamellipodia. As with most dynamic cellular systems, a collection of kinases, phosphatases, 

and adaptor proteins regulate the activity and localization of a few key effector proteins. p120 catenin 

(δ-catenin) binds and stabilizes cadherin at the plasma membrane. Membrane-bound and cytosolic 

tyrosine kinases phosphorylate β-catenin at weak or nascent junctions, while phosphatases remove 

added phosphates from β-catenin and δ-catenin at established junctions. Rho family GTPases modulate 

the availability and activation state of catenins and other essential adherens proteins. Together, this 

collection of structural proteins, enzymes, and adaptor proteins creates dynamic cell-cell junctions 

necessary for temporary associations during morphogenesis and maintains the integrity of complex 

tissues and structures following development. 

E-cadherin, an important component of adherens 

junctions, localizes to the plasma membrane. 

E-Cadherin (24E10) Rabbit mAb 

#3195: Confocal IF analysis of MCF7 

cells using #3195 (green, left) compared 

to an isotype control (right). Blue pseudocolor 

= DRAQ5 ® (fluorescent DNA dye). 

04 Cell Biology Tight Junctions 

Tight junctions are impermeable cell-cell junctions that form a continuous barrier to fluids across the 

epithelium and endothelium. They function in regulation of paracellular permeability and in the maintenance 

of cell polarity, blocking the movement of transmembrane proteins between the apical and the 

basolateral cell surfaces. The primary protein families composing tight junctions are claudin, occludins, 

and junctional adhesion molecules (JAMs) transmembrane proteins, which join the junctions to the 

cytoskeleton. Occludin is thought to be important in the assembly and maintenance of tight junctions. 

Differential phosphorylation of occludin at various residues may regulate its interaction with other tight 

junction proteins such as ZO-1. 

Differential expression of Claudin-1, a 

component of tight junctions, in IGROV-1 (high), 

A549 (moderate), and 293 (absent) cells 

Claudin-1 (D3H7C) Rabbit mAb #13995: WB analysis of extracts from IGROV-1, A549, 

and 293 cells using #13995 (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). 

kDa 

100 

80 

60 

50 

40 

30 

20 

50 

40 

1 2 3 

Lanes 

1. IGROV-1 

2. A549 

3. 293 

Claudin-1 

β-Actin 


Focal adhesions are the connections that form between a cell and the extracellular matrix (ECM) and are 

mediated primarily through integrins. Integrins are cell surface receptors that play a pivotal role in cell 

adhesion, migration, invasion, growth, and survival. Integrins are α/β heterodimeric proteins composed 

of one α and one β subunit. The integrin family contains at least 18 α and 8 β subunits that form 24 

known integrin pairs with distinct tissue distribution and overlapping ligand specificities. The intracellular 

tail of integrins interacts with cytoskeletal proteins vinculin, talin, and α-actinin, as well as numerous 

signaling molecules such as focal adhesion kinase (FAK). Activation of FAK by integrin clustering leads 

to autophosphorylation at Tyr397, which is a binding site for the Src family kinases PI3K and PLCγ. 

α/β Integrin Pairs 

αIIb 

chapter 04: Cell Biology 

Altered expression 

of Claudin-1 can be 

found in many types 

of cancer. 

Claudin-1 (D5H1D) XP ® Rabbit mAb 


human colon carcinoma (top) and human 

lung carcinoma (bottom) using #13255. 

α1 

α11 

α10 

β3 

αL 

α2 

α5 

β5 

α3 

β1 

αV 

αX β2 αD 

N-cadherin, a central component of adherens 

junctions, is up-regulated in many cancers. 

N-Cadherin (D4R1H) XP ® Rabbit mAb #13116: IHC analysis 

of paraffin-embedded human ovarian carcinoma using #13116. 

αE 

β7 

α4 

α9 

α6 

β4 

α8 

α7 β8 

β6 

αM 

P-cadherin, an adherens junction component 

expressed in epithelial cells and some cancers, 

localizes to the plasma membrane. 

P-Cadherin (C13F9) Rabbit mAb #2189: Confocal IF analysis of A-431 cells using 

#2189 (green). Actin filaments were labeled with Alexa Fluor ® 555 Phalloidin #8953 

(red). Blue pseudocolor = DRAQ5 ® (fluorescent DNA dye). 

On SDS-PAGE, Integrin α4 can migrate as a 150 kDa 

mature protein, a 140 kDa precursor protein, or as 

an 80 kDa or 70 kDa cleavage fragment. 

kDa 

200 

140 

100 

80 

60 

50 

40 

1 2 3 

Full length 

Integrin α4 

Cleaved 

C-terminal 

Integrin α4 

Integrin α4 (D2E1) XP ® Rabbit 

mAb #8440: WB analysis of extracts 

from various cell lines using #8440. 

Lanes 

1. Jurkat 

2. MOLT-4 

3. C6 


www.cellsignal.com/cstadhesion 107


Example of Integrin β3 expression in cancer 

Integrin β3 (D7X3P) XP ® Rabbit mAb #13166: IHC analysis of 

paraffin-embedded human papillary renal cell carcinoma using #13166. 

Vinculin is expressed in human breast ductal carcinoma in situ. 

Vinculin (E1E9V) XP ® Rabbit mAb #13901: IHC analysis of paraffinembedded 

human breast ductal carcinoma in situ using #13901. 

Epithelial-Mesenchymal Transition (EMT) 

EMT is an essential process during development whereby epithelial cells acquire mesenchymal, fibroblast-like 

properties and display reduced intracellular adhesion and increased motility. This is a critical 

feature of normal embryonic development (type I) and wound healing (type II), but it is also utilized by 

malignant epithelial tumors to spread beyond their origin (type III). This tightly regulated process is associated 

with a number of cellular and molecular events. EMT depends on a reduction in expression of 

several cell adhesion molecules. For example, E-cadherin is a critical component of adherens junctions 

and is considered an active suppressor of invasion and growth for many epithelial cancers. Research 

studies have shown that cancer cells typically downregulate expression of E-cadherin and upregulate 

expression of N-cadherin. This is referred to as the cadherin switch and is one of the hallmarks of EMT. 

Downregulation of E-cadherin expression occurs by binding of transcriptional repressor proteins such 

as Slug, Snail, and ZEB to the E-cadherin promoter region. 

Type III EMT: Cancer Invasion 

Invading Cancer Cells 


EMT 

Proliferating 

Tumor Cells 

Endothelium 

Extracellular 

Matrix 


Ciobanasu, C., Faivre, B., and Le Clainche, C. (2013) Eur. J. Cell Biol. 92, 339−348. • Dejana, E. and Orsenigo, F. (2013) 

J. Cell Sci. 126, 2545−2549. • Giannotta, M., Trani, M., and Dejana, E. (2013) Dev. Cell 26, 441−454. • Ivanov, A.I. and 

Naydenov, N.G. (2013) Int. Rev. Cell Mol. Biol. 303, 27−99. • Lamouille, S., Xu, J., and Derynck, R. (2014) Nat. Rev. Mol. 

Cell Biol. 15, 178−196. • Lampugnani, M.G. (2012) Cold Spring Harb. Perspect. Med. 2, a006528. • Runkle, E.A. and Mu, 

D. (2013) Cancer Lett. 337, 41−48. • Takeichi, M. (2014) Nat. Rev. Mol. Cell Biol. 15, 397−410. • Van Itallie, C.M. and 

Anderson, J.M. (2013) Tissue Barriers 1, e25247. • Zheng, H. and Kang, Y. (2014) Oncogene 33, 1755−1763. 

Commonly Studied Adhesion Targets 

Target M P 

ADAMTS1 • 

ADAM9 

• • 

ADAM10 

• 

Afadin 

• • 

Phospho-Afadin (Ser1718) • 

Ajuba 

• 

BSP II 

• 

Pan-Cadherin • • 

E-Cadherin • 

N-Cadherin • • 

OB-Cadherin • • 

P-Cadherin • • 

VE-Cadherin • • 

α-E-Catenin • • 

Phospho-α-E-Catenin • 

(Ser652) 

Phospho-α-E-Catenin • 

(Ser655/Thr658) 

α-N-Catenin • • 

γ-Catenin 

• 

Phospho-Catenin δ-1 • 

(Tyr228) 


(Ser252) 


(Ser320) 


(Tyr904) 

Catenin δ-1 

• 

CD54/ICAM-1 • 

Target M P 

CD102/ICAM-2 

CEA/CD66e 

Claudin-1 

Connexin 43 

Phospho-Connexin 43 

(Ser368) 

CYR61 

Phospho-Desmoplakin 

(Ser165/166) 

EpCAM 

FAK 

Phospho-FAK (Tyr397) 

Phospho-FAK (Tyr576/577) 

GIT-1 

GIT2 

Phospho-GIT2 (Tyr392) 


Hic-5 

ILK1 

βIG-H3 

Integrin α2b 

Integrin α4 

Integrin α5 

Integrin aV 

Integrin a6 

Integrin β1 

Integrin β3 

Integrin β4 

Integrin β5 




16, 99−107. 

Ardiani, A. et al. (2014) Vaccine-mediated immunotherapy 

directed against a transcription factor driving the metastatic 

process. Cancer Res. 74, 1945−1957. 

Reynies, A. et al. (2014) Molecular classification of malignant 

pleural mesothelioma: identification of a poor prognosis 

subgroup linked to the epithelial-to-mesenchymal transition. 


Wang, Y. et al. (2014) CUL4A induces epithelial-mesenchymal 

transition and promotes cancer metastasis by regulating 

ZEB1 expression. Cancer Res. 15, 520−531. 

Rajabi, H. et al. (2014) MUC1-C oncoprotein activates the 

ZEB1/miR-200c regulatory loop and epithelial-mesenchymal 

transition. Oncogene 33, 1680−1689. 

Kao, C.J. et al. (2014) miR-30 as a tumor suppressor 

connects EGF/Src signal to ERG and EMT. Oncogene 33, 

2495−2503. 

Truong, H.H. et al. (2014) beta1 integrin inhibition elicits a 

prometastatic switch through the TGFbeta-miR-200-ZEB 

network in E-cadherin-positive triple-negative breast cancer. 

Sci. Signal. 7, ra15. 

Geng, Y. et al. (2014) Insulin receptor substrate 1/2 (IRS1/2) 

regulates Wnt/beta-catenin signaling through blocking 

autophagic degradation of dishevelled2. J. Biol Chem. 289, 

11230−11241. 

Ludwig, K. et al. (2013) Colon cancer cells adopt an invasive 

phenotype without mesenchymal transition in 3-D but not 

2-D culture upon combined stimulation with EGF and crypt 

growth factors. BMC Cancer 13, 250−257. 

• 

• 

• • 

• 

• 

• 

• 

• • 

• • 

• • 

• 

• 

• • 

• 

• 

• 

• • 

• • 

• 

• • 

• 

• 

• 

• • 

• • 

• 

• • 

Target M P 

LPP 

Lyric/Metadherin 

Maspin 

Mesothelin 

MMP-2 

MMP-7 

MMP-9 

MUC1 

NCAM (CD56) 

α-Parvin 

Paxillin 

Phospho-Paxillin (Tyr118) 

PSA/KLK3 

RECK 

Renin 

Talin-1 

Phospho-Talin (Ser425) 

TIMP1 

TIMP2 

TIMP3 

uPAR 

Vinculin 

ZO-1 

ZO-2 

ZO-3 

Zyxin 

Phospho-Zyxin 

(Ser142/143) 

• 

• 

• 

• 

• • 

• 

• • 

• 

• • 

• • 

• • 

• 

• 

• 

• 

• 

• • 

• 

• 

• 

• • 

• • 

• • 

• 

• 

• 

• • 

Chiyomaru, T. et al. (2014) Long non-coding RNA HOTAIR is 

targeted and regulated by miR-141 in human cancer cells. 

J. Biol. Chem. 289, 12550−12565. 

Akalay, I. et al. (2013) Epithelial-to-mesenchymal transition 

and autophagy induction in breast carcinoma promote 

escape from T-cell-mediated lysis. Cancer Res. 73, 

2418−2427. 

Song, S. et al. (2013) Loss of TGF-beta adaptor beta2SP 

activates notch signaling and SOX9 expression in esophageal 

adenocarcinoma. Cancer Res. 73, 2159−2169. 

Shi, Z. et al. (2013) mTOR signaling feedback modulates 

mammary epithelial differentiation and restrains invasion 

downstream of PTEN loss. Cancer Res. 73, 5218−5231. 

Dasgupta, S. et al. (2013) Novel role of MDA-9/syntenin in 

regulating urothelial cell proliferation by modulating EGFR 

signaling. Clin Cancer Res. 19, 4621−4633. 

Smith, N.R. et al. (2013) Tumor stromal architecture can 

define the intrinsic tumor response to VEGF-targeted therapy. 

Clin Cancer Res. 19, 6943−6956. 

Domyan, E.T. et al. (2013) Roundabout receptors are critical 

for foregut separation from the body wall. Dev. Cell 24, 

52−63. 

Yu, Y.H. et al. (2013) MiR-520h-mediated FOXC2 regulation is 

critical for inhibition of lung cancer progression by resveratrol. 

Oncogene 3, 431−443. 

Rizvi, I. et al. (2013) Flow induces epithelial-mesenchymal 

transition, cellular heterogeneity and biomarker modulation 

in 3D ovarian cancer nodules. Proc. Natl. Acad. Sci. USA 

110, 1974−1983. 

Gumireddy, K. et al. (2013) Identification of a long noncoding 

RNA-associated RNP complex regulating metastasis 

at the translational step. EMBO J. 32, 2672−2684. 



nodes within adhesion signaling 

pathways and are commonly 

studied in adhesion research. Primary 








163 


CST antibodies for E-Cadherin 




www.cellsignal.com/cstadhesion 109


Adherens Junction Dynamics 

Cytoplasm 

Extracellular 

Space 

Cytoplasm 

Adherens 

Junction 

Remodeling 

VE-Cadherin 

Occludin 

MLC 

Nucleus 

Cadherin 

IQGAP 

WASP 

Arp2/3 

Formation 

Fyn Gβ1 δ-cat 

p190 RhoA 

δ-cat 

SH2D1A 

Cytoplasm 

c-Cbl 

α-cat β-cat 

LAMTOR1 

Rac 

PRK1 

Par3/6 aPKC 

RhoA 

ARHGAP1 

δ-cat 

Ect2 Cortactin 

cdc42 mDIA 

Centralspindlin 

Complex 

Increased 

Actomycin 

Contractility 

• Actin Nucleation 

• Actin Dynamics 

Vinculin 

ROCK 

δ-cat 

Kaiso 

IRSp53 

β-cat 

β-cat 

LEF/TCF 

Maintenance 

Cadherin 

Rac 

IQGAP 

β-cat 

α-cat 

PI3K 

Akt 

IQGAP 

PTPµ 

δ-cat 

Rac 

β-cat 

δ-cat 

α-cat β-cat 

Vinculin 

Tiam1 

CK2 

Src 

• Anti-Apoptotic Signals 

• Apoptosis 

• Angiogenesis 

Wnt 

Signaling 

PTP1B 

Cadherin 

Recycling 

Ajuba 

α-cat 

Arp2/3 

Cadherin 

Degradation 

Cell Growth & Differentiation 

Nectin 

Afadin 

α-cat 

γ-cat 

Formin 

α-cat 

RaIA 

α 

Nectin 

Clathrin 

Afadin 

Rap1 

α 

Src 

Disassembly 

Axin1 

β-cat 

E3 

Actin bundles 

Actin 

Disassembly 

Caveolin/Clathrin 

Phagosome 

α 

Caveolin 

Ub UbUb 

β-cat 

α-actinin 

Cadherin Endocytosis 

WASP 

Cip4 

APC 

GSK-3 

β-cat 

APC 

Ub 

Ub 

Ub 

Ub 

β-cat 

β-catenin 

Degradation 

Adherens junctions are dynamic structures that form, strengthen and spread, degrade, and then re-form as their associated proteins create ephemeral connections with 

counterparts from adjacent cells. This view updates the traditional model of a stable complex composed of cadherin, β-catenin, and α-catenin bound to the actin cytoskeleton. 

Although cadherin does exist in a complex with β-catenin and α-catenin, this cadherin-catenin complex does not associate with the actin cytoskeleton. α-catenin does not 

directly anchor cell adhesion proteins to the actin cytoskeleton but acts as a regulatory protein to control actin filament dynamics. 

Monomeric α-catenin binds β-catenin at adherens junctions and upon release forms α-catenin dimers that promote actin bundle formation. The transition from branched actin 

networks to bundled actin filaments correlates with the creation of mature, strong adherens junctions and a decrease in membrane lamellipodia. The connection between cell 

junctions and the cytoskeleton may be more dynamic than originally considered and may rely on multiple, weak associations between the cadherin-catenin complex and the 

actin cytoskeleton or rely on other membrane-associated proteins (i.e. nectin and afadin). 

As with most dynamic cellular systems, a collection of kinases, phosphatases, and adaptor proteins regulate the activity and localization of a few key effector proteins. 

δ-catenin (p120 catenin) binds and stabilizes cadherin at the plasma membrane. Membrane bound and cytosolic tyrosine kinases phosphorylate β-catenin at weak or nascent 

junctions, while phosphatases remove added phosphates from β-catenin and δ-catenin at established junctions. Rho family GTPases modulate the availability and activation 

state of catenins and other essential adherens proteins. Together, this collection of structural proteins, enzymes, and adaptor proteins creates dynamic cell-cell junctions 

necessary for temporary associations during morphogenesis and maintains the integrity of complex tissues and structures following development. 


Baum, B. and Georgiou, M. (2011) J. Cell Biol. 192, 907–917. • Baumann, K. (2013) Nat. Rev. Mol. Cell Biol. 14, 68. • Citi, S., Spadaro, D., Schneider, Y., Stutz, J., and 

Pulimeno, P. (2011) Mol. Membr. Biol. 28, 427–444. • Harris, T.J. and Tepass, U. (2010) Nat. Rev. Mol. Cell Biol. 11, 502–514. • Niessen, C.M. and Gottardi, C.J. (2008) 

Biochim. Biophys. Acta 1778, 562–571. • Pieters, T., van Roy, F., and van Hengel, J. (2012) Front. Biosci. 17, 1669–1694. • Yonemura, S. (2011) Curr. Opin. Cell Biol. 

23, 515–522. 

Cytoskeletal Regulation 

The cytoskeleton consists of three types of cytosolic fibers: microtubules, microfilaments (actin 

filaments), and intermediate filaments. Cytoskeletal signaling regulates several important cellular 

processes such as cell division, adhesion, polarity, migration, and movement through cilia and flagella. 

Microtubules 

Microtubules are composed of globular tubulin subunits, with α/β-tubulin heterodimers forming the 

tubulin subunit common to all eukaryotic cells. γ-tubulin is required to nucleate polymerization of 

tubulin subunits to form microtubule polymers. Many cell movements are mediated by microtubule 

action, including the beating of cilia and flagella, cytoplasmic transport of membrane vesicles, and 

nerve-cell axon migration. Microtubules also play a critical role in spindle assembly during mitosis/ 

meiosis and are responsible for chromosome alignment during metaphase. Microtubules form the 9+2 

structure of the centriole, a critical component of the centrosome that acts as a microtubule-organizing 

center (MTOC) and plays a role in cell polarity. Because of their role in mitosis, microtubules have been 

targets of chemotherapy in cancer. Microtubules continuously undergo a process of dynamic instability, 

whereby microtubule polymerization on the plus end competes with depolymerization at the minus end. 

This process is regulated by several signaling molecules including stathmin, diap1/2, tau, and the Rho 

family of small GTPases. 

Stathmin is a microtubule destabilizing 

protein phosphorylated at Ser38 in cells 

synchronized in mitosis. 

Phospho-Stathmin (Ser38) (D19H10) Rabbit mAb #4191: WB analysis 

of extracts from HT-29 and U-2 OS cells, untreated or synchronized in 

mitosis, using #4191. Mitotic synchrony was performed by using either a 

thymidine block followed by release into nocodazole (100 ng/ml, 24 hr) or 

using Docetaxel #9886 (100 ng/ml, 24 hr). 

kDa 

60 

50 

40 

30 

20 

10 

1 

2 3 

– + – + – – 

– – – – – + 

Lanes 

1. HT-29 

2. U-2 OS 

3. HT-29 

Phospho- 

Stathmin 

(Ser38) 

Thymidine/ 

Nocodazole 

Microfilaments 

Microfilaments are major structural components of the cytoskeleton and consist of fibrous polymers of 

actin, called F-actin. Microfilaments are important for changes in cell shape, migration, proliferation, 

and survival. Regulation of the actin cytoskeleton begins with signaling through G protein-coupled 

receptors (GPCRs), integrins, receptor tyrosine kinases (RTKs), and numerous other specialized receptors 

such as the semaphorin 1a receptor PlexinA. These receptors initiate a large number of signaling 

cascades that include the Rho family of small GTPases (Rho, Rac, and Cdc42) and their activators, 

guanine nucleotide exchange factors (GEFs) and their downstream protein kinase effectors (ROCK and 

PAK), as well as through direct binding of the GTPases to several actin regulatory proteins (cortactin, 

diap1/2, WAVE, and WASP). These cascades converge on proteins that directly regulate the behavior 

and organization of the actin cytoskeleton, including actin interacting regulatory proteins such as 

cofilin, ADF, Arp2/3 complex, Ena/VASP, profilin, and gelsolin. 

Small GTPases 

P 1 

Upstream signals 

Rho 

GTP 

GTP 

Docetaxel 


α/β tubulin heterodimers, 

the building 

blocks of microtubules, 

are found throughout 

the cytoplasm. 

β-Tubulin (9F3) Rabbit mAb (Alexa 

Fluor ® 488 Conjugate) #3623: Confocal 

IF analysis of HeLa cells using #3623 



COS-7 cells express 

cofilin phosphorylated 

at Ser3, a modification 

known to inhibit cofilin 

activity. 

kDa 

60 

50 

40 

30 

20 

10 

60 

50 

40 

30 

Phospho- 

Cofilin (Ser3) 

GTPase-activating 

protein 

GTPase 

cycle 

Guanine nucleotide 

exchange factor 

20 

Cofilin 

© 2008–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Kris DeMali of The University of Iowa Carver College of Medicine, Iowa City, IA for reviewing this diagram. 

H 2O 

Rho 

GTP 

Downstream effectors 

GDP 

10 

– + 

λ-phosphatase 

Phospho-Cofilin (Ser3) (77G2) Rabbit 

mAb #3313: WB analysis of COS-7 

cells, untreated or λ phosphatase-treated, 

using #3313 (upper) or Cofilin Antiobdy 

#3312 (lower). 


Actin cytoskeleton 

www.cellsignal.com/cstcytoskeletal 111


Intermediate filament 

protein desmin is expressed 

in muscle cells. 

Desmin (D93F5) XP ® Rabbit mAb 

#5332: Confocal IF analysis of mouse 

heart tissue using #5332 (green). Blue 


DNA dye). 

Vimentin is an 

intermediate filament 

protein expressed in 

connective tissue 

and other cells of 

mesenchymal origin. 

Nonmuscle myosin IIb 

heavy chain isoform 

colocalizes with actin. 

Myosin IIb (D8H8) XP ® Rabbit mAb 

#8824: Confocal IF analysis of COS-7 

cells, showing Myosin (A), Actin (B), 

and merged (C), using #8824 (green). 

Actin filaments were labeled with DY-554 



Intermediate Filaments 

The major types of intermediate filaments are distinguished by their cell-specific expression. 

Classes of 

Intermediate 

Filaments 

Class Protein 

Expression 

I Acidic keratins Epithelial cells 

II Basic keratins Epithelial cells 

III Desmin, GFAP, vimentin Muscle, Glial cells, Mesenchymal cells 

IV Neurofilaments (NFL, NFM and NFH) Neurons 

V Lamins Nucleus 

Members of this group contain a globular N-terminal head domain, a central α−helical rod domain, and 

a variable C-terminal tail. Intermediate filaments provide structural support for the cell, act as anchorage 

points for organelles and molecular motors, and function as stress proteins that provide protection 

from intrinsic and environmental stresses. Intermediate filaments can be regulated through several 

mechanisms including phosphorylation, which affects their activity or ability to assemble with other 

intermediate filament-interacting proteins. 

A 

A 


B 

B 

Vimentin (D21H3) XP ® Rabbit mAb #5741: 

IHC analysis of paraffin-embedded mouse 

colon (A) using #5741. Confocal IF analysis 

of SNB19 cells (B) using #5741 (green). Blue 


DNA dye). 

Myosins 

Myosins are a large superfamily of actin-binding motor proteins that use ATP hydrolysis to generate 

motility. Myosins are categorized in over 25 classes. Of these, class II myosins, known as conventional 

myosins, comprise the largest class and contain myosin proteins specific to skeletal, cardiac, and 

smooth muscle as well as nonmuscle isoforms. Nonmuscle myosin is essential to cell motility, cell 

division, migration, adhesion, and polarity. The holoenzyme consists of two identical heavy chains and 

two sets of light chains. The light chains (MLCs) regulate myosin II activity and stability and exist in 

many isoforms with varying tissue distribution. The heavy chains (NMHCs) are encoded by three genes, 

MYH9, MYH10, and MYH14, which generate three different nonmuscle myosin II isoforms, IIa, IIb, and 

IIc, respectively. While all three isoforms perform the same enzymatic tasks, binding to and contracting 

actin filaments coupled to ATP hydrolysis, their cellular functions do not appear to be redundant and 

they have different subcellular distributions. 


Blanchoin, L., Boujemaa-Paterski, R., Sykes, C., et al. (2014) Physiol. Rev. 94, 235−263. • Chircop, M. (2014) Small GTPases 

5, e29770. • Chung, B.M., Rotty, J.D., and Coulombe, P.A. (2013) Curr. Opin. Cell Biol. 25, 600−612. • Gurel, P.S., Hatch, 

A.L., and Higgs, H.N. (2014) Curr. Biol. 24, 660−672. • Julian, L. and Olson, M.F. (2014) Small GTPases 5, e29846. • Kull, 

F.J. and Endow, S.A. (2013) J. Cell Sci. 126, 9−19. • Parker, A.L., Kavallaris, M., and McCarroll, J.A. (2014) Front. Oncol. 4, 

153. • Snider, N.T. and Omary, M.B. (2014) Nat. Rev. Mol. Cell Biol. 15, 163−177. • Wojnacki, J., Quassollo, G., Marzolo, 

M.P., et al. (2014) Small GTPases 5, e28430. 

C 

Commonly Studied Cytoskeletal Regulation Targets 

Target M P Target M P Target M P 

14-3-3 Family • Diap1 

• Keratin 8/18 • 

14-3-3 β/α • Diap2 

• Keratin 17 • 

14-3-3 γ • DOCK180 • Phospho-Keratin 17 (Ser44) • 

14-3-3 ε • DRP1 

• Keratin 17/19 • 

14-3-3 ζ/δ • Phospho-DRP1 (Ser616) • • Keratin 18 • 

14-3-3 η • • Phospho-DRP1 (Ser637) • • Keratin 19 • 

14-3-3 τ • DSG2 

• Keratin 20 • 

Phospho-Ack1 (Tyr284) • EB-1 

• Phospho-KIF1B (Ser1487) • 

Pan-Actin • • Emerin 

• • KIFC1 

• 

α-Actinin • • EML4 

• • KIF3A 

• 

Afadin 

• • EPAC1 

• KIF3B 

• 

Annexin V 

• EPAC2 

• Kinectin 1 • • 

Phospho-AP2M1 (Thr156) • • EpCAM 

• • Lamin A/C • • 

Phospho-ARHGAP42 • EPLIN 

• Phospho-Lamin A/C (Ser22) • • 

(Tyr376) 

Erlin-1 

Cool2/αPix • 

• Lamin B1 • 

Erlin-2 

Cool1/βPix 

• 

• Lamin B2 • 

EVL 

ARP2 

• • 

• LAMP1 

• 

Ezrin 

ARP3 

• 

• LASP1 

• 

Phospho-Ezrin (Tyr353) 

Atlastin-1 • 

• • LCP1 

• • 

Ezrin/Moesin/Radixin 

β-Actin 

• • 

• Phospho-LCP1 (Tyr28) • 

Phospho-Ezrin (Thr567)/ 


• • LIMK1 

• 

Radixin (Thr564)/Moesin 

(Ser320) 

(Thr558) 

Phospho-LIMK1 (Thr508)/ • 

β2-Chimerin 

LIMK2 (Thr505) 

• Fascin 

• LIMK2 

Caldesmon-1 • • 

• 

Fer 

• LLGL1 

Caveolin-1 • • 

• 

Fes 

• LMAN1 

Phospho-Caveolin-1 (Tyr14) • 

• 

Fibrillarin • Phospho-MARK Family 

Caveolin-2 • 

• 

Filamin A 

• (Activation Loop) 

CD2AP 

• Phospho-Filamin A 

MARK1 

• 

• 

CDC37 

(Ser2152) 

• • 

MARK2 

• 

Filamin B 

Phospho-CDC37 (Ser13) • 

• • MARK3 

• 

Filamin C 

Cdc42 

• • 

• MARK4 

• 

Flotillin-1 

CD71 

• 

• MCF2/Dbl 

• 

Flotillin-2 

CdGAP 

• 

• • Moesin 

• 

FYVE-CENT 

Centrin-2 

• 

• M-RIP 

• 

GEF-H1 

Chronophin/PDXP • 

• • MTSS1 

• 

Gelsolin 

CIN85 

• 

• • Myosin IIa 

• 


Claudin-1 • • 

• Phospho-Myosin IIa • 

GM130 

CLIP1/CLIP170 • 

• • (Ser1943) 

Golgin-97 

Myosin IIb 

Cofilin 

• • 

• 

• • 

HEF1/NEDD9 

Myosin IIc 

Phospho-Cofilin (Ser3) • • 

• 

• • 

Importin β1 

Myosin Va 

Cortactin 

• 

• 

• 

IQGAP1 

Myosin VI 

Phospho-Cortactin (Tyr421) • 

• 

• 

IQGAP2 

Myosin Light Chain 2 

CrkII 

• 

• 

• • 

Integrin α2b 

Phospho-Myosin Light Chain 

Phospho-CrkII (Tyr221) • 

• 

• • 

2 (Ser19) 

Pan-Keratin 

Dab2 

• • 

• Phospho-Myosin Light Chain • 

Keratin 7 

2 (Thr18/Ser19) 

Desmin 

• • 

• 


These protein targets represent key nodes 

within cytoskeletal regulation signaling 

pathways and are commonly studied in 

cytoskeletal regulation research. Primary 








www.cellsignal.com/cstcytoskeletal 113


104 


CST antibodies for β-tubulin 





Harikumar, K.B. et al. (2014) 

K63-linked polyubiquitination of 

transcription factor IRF1 is essential 

for IL-1-induced production of 

chemokines CXCL10 and CCL5. Nat. 

Immunol. 15, 231−238. 

Kim, D.K. et al. (2014) Inverse agonist 

of estrogen-related receptor gamma 

controls Salmonella typhimurium 

infection by modulating host iron homeostasis. 

Nat. Med. 20, 419−424. 

Bohrer, L.R. et al. (2014) Activation 

of the FGFR-STAT3 pathway in breast 

cancer cells induces a hyaluronan-rich 

microenvironment that licenses tumor 

formation. Cancer Res. 74, 374−386. 

Lucas, C.L. et al. (2014) Dominantactivating 

germline mutations in the 

gene encoding the PI(3)K catalytic 

subunit p110delta result in T cell 

senescence and human immunodeficiency. 

Nat. Immunol. 15, 88−97. 

Zhu, Y. et al. (2014) Role of tumor 

necrosis factor alpha-induced protein 

1 in paclitaxel resistance. Oncogene 

33, 3246−3255. 

Sun, X. et al. (2014) p27 Protein 

Protects Metabolically Stressed 

Cardiomyocytes from Apoptosis by 

Promoting Autophagy. J. Biol. Chem. 

289, 16924−16935. 

Barry, E.R. et al. (2013) Restriction of 

intestinal stem cell expansion and the 

regenerative response by YAP. Nature 

493, 106−110. 

Raj, V.S .et al. (2013) Dipeptidyl 

peptidase 4 is a functional receptor 

for the emerging human coronavirus- 

EMC. Nature 495, 251−254. 

Manzanillo, P.S. et al. (2013) The 

ubiquitin ligase parkin mediates 

resistance to intracellular pathogens. 

Nature 501, 512−616. 

Chowdhury, S. et al. (2013) Flavoneresistant 

Leishmania donovani 

Overexpresses LdMRP2 Transporter in 

the Parasite and Activates Host MRP2 

on Macrophages to Circumvent the 

Flavone-mediated Cell Death. J. Biol. 

Chem. 289, 16129−16147. 

Wang, D. et al. (2013) MicroRNA-205 

controls neonatal expansion of 

skin stem cells by modulating the 

PI(3)K pathway. Nat. Cell Biol. 15, 

1153−1163. 

Target M P Target M P Target M P 

Myosin Light Chain 2v • PREX1 

• Sec31A 

• 

(Cardiac Isoform) 

Profilin-1 

MYPT1 

• • SPAK 

• 

• • 

PTP4A3 

Phospho-MYPT1 (Ser507) 

• SSH1 

• 

• 

PVR/CD155 

Phospho-MYPT1 (Ser668) 

• Stathmin 

• 

• 

R-Ras 

Phospho-MYPT1 (Thr696) 

• • Phospho-Stathmin (Ser16) • 

• 

Rac1/Cdc42 

Phospho-MYPT1 (Thr853) 

• Phospho-Stathmin (Ser38) • • 

• 

Phospho-Rac1/cdc42 

Na,K-ATPase 

• Talin-1 

• 

• (Ser71) 

Phospho-Talin (Ser425) 

Phospho-Na,K-ATPase a1 • • Rac1/Rac2/Rac3 

• • 

• 

(Tyr10) 

TCTP 

RACK1 

• • 

Phospho-Na,K-ATPase a1 

• • 

• 

Phospho-TCTP (Ser46) 

(Ser16) 

Radixin 

• 

• 

Phospho-Na,K-ATPase a1 

Tensin 2 

• RalA 

• 

(Ser23) 

• • 

TESK1 

N-WASP 

RalB 

• 

• • 

• 

Troponin T (Cardiac) 

NCK1 

RalBP1 

• 

• 

• • 

Tropomyosin-1 

NTF2 

Ran 

• 

• 

• 

Tropomyosin-1/3 

NUP88 

RanBP1 

• 

• 

• 

Troponin I 

NUP98 

Phospho-RanBP3 (Ser58) 

• • 

• • 

• 

Phospho-Troponin I (Cardiac) 

OSR1 

Rap1A/Rap1B 

• 

• 

• • (Ser23/24) 

PAK1/2/3 

Rap1B 

• 

• α-Tubulin • • 

PAK1 

RasGRP3 

• 

• Acetyl-α-Tubulin (Lys40) • • 

Phospho-PAK1 (Ser144)/ 

RCC1 

• 

• • α/β-Tubulin 

• 

PAK2 (Ser141) 

Phospho-RCC1 (Ser11) • • β-Tubulin • • 

Phospho-PAK1 • RCC2 

(Ser199/204)/PAK2 

• • γ-Tubulin 

• 

(Ser192/197) 

Phospho-REPS1 (Ser709) • Twinfilin-1 • • 

Phospho-PAK1 (Thr423)/ • RhoA 

PAK2 (Thr402) 

• VASP 

• • 

PAK2 

• • 

RhoB 

• Phospho-VASP (Ser157) • 

Phospho-PAK2 (Ser20) • 

RhoC 

• Phospho-VASP (Ser239) • 

PAK3 

• 

RhoE 

• Vav1 

• • 

PAK4 

• 

p190-A RhoGAP • • Vav2 

• 

Phospho-PAK4 (Ser474)/ 

p190-B RhoGAP 

• 

• Vav3 

• 

PAK5 (Ser602)/PAK6 

RhoGDI 

(Ser560) 

• Villin-1 

• 

PAR2 

• 

p115 RhoGEF • Vimentin • • 

PCM-1 

• 

ROCK1 

• Phospho-Vimentin (Ser39) • 

PDLIM2 

• 

ROCK2 

• • Phospho-Vimentin (Ser56) • • 

Podoplanin • 

SCAI 

• Phospho-Vimentin (Ser83) • • 

PKG-1 

• 

Sec23A 

• WASP 

• • 

PKG-1α 

• 

Sec24A 

• WAVE-2 • 

Plectin-1 • • 

Sec24B 

• • WAVE-3 

• 

PRC1 

• 

Sec24C 

• ZO-1 

• • 

Sec24D 

• 

Antibody Validation Principles 

Please visit our website to learn more about what Antibody Validation means at Cell Signaling Technology. 


Regulation of Actin Dynamics 

F-actin 

Bundles/ 

Meshworks 


PlexinA 

PI3K PLCγ PIP 2 

G 

Src 

PI3K 

i G 

Grb2 Nck 

q G s 

FAK PIP 

Paxillin 

3 Ca 2+ 

Mical 

CaM PKCα 

PLCβ 

NADPH 

GRB2 

PAK1 WASP 

Fascin 

PIP PI3Kα 

NADP 

3 

GEF 

Filopodia 

Ena 

p130 GIT1/2 

Ras 

PKA 

VASP 

PI3K 

PIP 3 

PI3K 

PIX 

Cofilin 

MEKK 

RhoGEF 

TIAM1 

PIX 

Vav 

MEKK 

Degradation 

Crk 

Rac 

cdc42 PAK1 MEK 

Rho 

Cortactin 

PAK1 

mDIA 

ROCK Filamin 

WAVE 

WASP 

ROCK 

PI5K 

MLCK 

MBS 

Arp2/3 

Ca 2+ 

PIP 2 

IP 3 CaM 

LIMK MLCK mDIA Arp2/3 

Filamin 

PKD1 

Profilin 

14-3-3ζ 

Calcineurin SSH 

MLC 

Severed 

Filaments 

Sema1a 

Gelsolin 

Lamellipodia 

Integrin 

Receptor 

CIN 

Hsp90 

RTK 

Cofilin ADF Filopodia 

Barbed-end 

CapZ Cappers 

Pointed-end 

Tmod Cappers 

Actin 

Polymerization 

Tropomyosins 

Lamellipodia 

Stress Fibers 

Signaling to the cytoskeleton through G protein-coupled receptors (GPCRs), integrins, receptor tyrosine kinases (RTKs), and numerous other specialized receptors, such as the 

semaphorin 1a receptor PlexinA, can lead to diverse effects on cell activity, including changes in cell shape, migration, proliferation, and survival. Integrins, in conjuction with 

other components of focal adhesion complexes, serve as the link between the extracellular matrix and cytoskeleton in many cell types. Integrin activation leads to activation of 

focal adhesion kinase (FAK) and Src kinase, resulting in phosphorylation of other FA components such as paxillin and the Crk-associated substrate p130 Cas, as well as the 

recruitment of signaling adapter proteins. 

Intracellular regulation of the cell’s response to external cues occurs through a large number of signaling cascades that include the Rho family of small GTPases (Rho, Rac, 

and Cdc42) and their activators, guanine nucleotide exchange factors (GEFs), their downstream protein kinase effectors, including Rho-kinase/ROCK and p21 activated 

kinase (PAK), as well as through direct binding of the GTPases to several actin regulatory proteins, such as cortactin, mDia, WAVE, and WASP. These cascades converge on 

proteins that directly regulate the behavior and organization of the actin cytoskeleton, including actin interacting regulatory proteins such as cofilin, Arp2/3 complex, Ena/VASP, 

forminins, profilin, and gelsolin. Signaling through different pathways can lead to the formation of distinct actin-dependent structures whose coordinated assembly/disassembly 

is important for directed cell migration and other cellular behaviors. Migration is also regulated by signaling to myosin, which participates in leading edge actin dynamics 

and enables retraction of the rear of the cells. Tropomyosins stabilize F-actin by preventing binding of severing and dynamizing factors. Some tropomyosins may also enhance 

filament dynamics. Dynamic actin is required for most cellular actin-dependent processes; inhibiting actin assembly and preventing actin disassembly are equally inhibitory to 

most behaviors. 

Aberrant control of cytoskeletal signaling, which can result in a disconnection between extracellular stimuli and cellular responses, is often seen in immune pathologies, 

developmental defects, and cancer. 


Bernstein, B.W. and Bamburg, J.R. (2010) Trends Cell Biol. 20, 187–195. • Lee, S.H. and Dominguez, R. (2010) Mol. Cells 29, 311–325. • Levayer, R. and Lecuit, T. 

(2012) Trends Cell Biol. 22, 61–81. • Poukkula, M., Kremneva, E., Serlachius, M., and Lappalainen, P. (2011) Cytoskeleton (Hoboken) 68, 471–490. • Ridley, A.J. (2011) 

Cell 145, 1012–1022. • Rottner, K. and Stradal, T.E. (2011) Curr. Opin. Cell Biol. 23, 569–578. 

© 2008–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. James Bamburg, Colorado State University, Fort Collins, CO for reviewing this diagram. 

G 13 

GPCR 



115


Regulation of Microtubule Dynamics 

Spred1 

TESK 

Cofilin 

LIMK 

TPPP 

Par1 

TAOK 

MARK 

PIP 3 

LL5β 

Rac1 

cdc42 

Par6 

Rac1 

Tau 

MT Stability 

LRP 

Par3 

mDIA 

Delivery to 

MT Plus Ends 

Src 

Tiam1 

aPKC 

Rho 

mDIA 

APC 

EB1 

CLIP 

c-Abl 

Trio 

Gα q/o 

Wnt 

MARK2 

Dvl 

MT 

Polymerization 

CLASP 

EB1 

CLIP 

Plus End 

Rho 

Plus End 

Proteins 

CLIP 

CLASP 

EB1 

APC 

mDIA1 

Erk 

MAPKAPK 

GSK-3β 

CRMP2 

Gα 

CLIP 

MAP1b 

XMAP215 

PI3K 

Akt PIP 3 

PTEN ROCK 

ICIS 

MCAK 

RTK 

Cdk1 

+ end Growth 

Promoting +/- end 

Destabilizing 

“Dynamic 

Microtubules” 


Rho 

MAP1b 

CaMK 

Aurora B 

Neurotrophins 

Actin Filaments 

Minus End 

PKA 

Rho 

PAK 

Stathmin 

MT Catastrophe 

RhoGEF 

Erk 

Stat3 

Protein Folding and 

VesIcle Trafficking 

Newly synthesized proteins must be properly folded and then directed to their correct subcellular locations 

in order to perform their biological functions. This highly regulated process includes molecular 

chaperone proteins that assist with proper folding and vesicle trafficking proteins that regulate delivery 

of cargo throughout the cell. 

Protein Folding: Heat Shock Proteins 

Heat Shock Proteins (HSPs) form seven families (small HSPs (sHSPs), HSP10, 40, 60, 70, 90, and 

100) of molecular chaperone proteins that play a central role in the cellular resistance to stress and 

actin organization. They are involved in the proper folding of proteins and the recognition and refolding 

of misfolded proteins. HSP expression is induced by a variety of environmental stresses, including heat, 

hypoxia, nutrient deficiency, free radicals, toxins, ischemia, and UV radiation. HSP27 is a member of 

the sHSP family. It is phosphorylated at Ser15, Ser78, and Ser82 by MAPKAPK-2 as a result of the 

activation of the p38 MAP kinase pathway. Phosphorylation and increased concentration of HSP27 has 

been implicated in actin polymerization and reorganization. HSP70 and HSP90 interact with unfolded 

proteins to prevent irreversible aggregation and catalyze the refolding of their substrates in an ATP- and 

co-chaperone-dependent manner. HSP70 has a broad range of substrates including newly synthesized 

and denatured proteins, while HSP90 tends to have a more limited subset of substrates, most of which 

are signaling molecules. HSP70 and HSP90 are also essential for the maturation and inactivation of 

nuclear hormones and other signaling molecules. 

Activation of the p38 MAPK pathway 

by UV or anisomycin results in phosphorylation 

of HSP27 at Ser82. 

Phospho-HSP27 (Ser82) (D1H2) XP ® Rabbit mAb #9709: WB 

analysis of extracts from HeLa or HT-29 cells, untreated (-) or treated 

(+) with either UV (40 mJ/cm 2 with 30 min recovery) or anisomycin 

(25 μg/ml, 30 min), using #9709. 

kDa 

80 

60 

50 

40 

30 

20 

1 

– + – – – 

+ + 

– – – 

2 

Cells 

1. HeLa 

Phospho- 

HSP27 

(Ser82) 

UV 

Anisomycin 

2. HT-29 


HSP60, an important 

chaperone for folding 

key mitochondrial 

proteins, is expressed 

in A-204 cells. 

HSP60 (D6F1) XP ® Rabbit mAb 

#12165: Confocal IF analysis of A-204 





Microtubules are required for the establishment of cell polarity, polarized migration of cells, intracellular vesicle transport, and chromosomal segregation in mitosis. Microtubules 

(MTs) are nonequilibrium polymers of α/β-tubulin heterodimers, in which GTP hydrolysis on the β-tubulin subunit occurs following assembly. Most microtubules are 

nucleated from organizing centers. The most prevalent microtubule behavior is dynamic instability, a process of slow plus end growth coupled with rapid depolymerization 

(“catastrophe”) and subsequent rescue. Although microtubule minus ends show dynamic instability, albeit at a lower rate than the plus ends, the minus ends are usually 

capped and anchored at MT organizing centers and thus often do not participate in microtubule dynamics. 

Maintaining a balance between dynamically unstable and stable microtubules is regulated in large part by proteins that bind either tubulin dimers or assembled microtubules. 

Proteins that bind tubulin dimers include stathmin, which sequesters tubulin and enhances MT dynamics by increasing catastrophe frequency, and collapsin response mediator 

protein (CRMP2), which increases MT growth rate by promoting addition of tubulin dimers onto microtubule plus ends. Other proteins that associate with assembled MTs include 

those that bundle MTs (e.g. MAP1c), those that stabilize MTs (e.g. tau), and those that maintain MTs in a dynamic state (MAP1b). A major signaling pathway that regulates MT 

dynamics involves GSK-3β, a kinase typically active under basal growth conditions but locally inactive in response to signals that enhance MT growth and dynamics. 

In addition to the above factors, many MT motor proteins, and even non-motor proteins, aid in the dynamics of MTs. Proteins such as Xenopus microtubule associated protein 

215 (XMAP215), promote MT assembly through binding to tubulin dimer to facilitate its incorporation in the growing plus end. XMAP215 also may compete with some of the 

MT plus end binding proteins (+TIPS), of which the end binding protein EB1 appears to be the master organizer. Complexes between the adenomateous polyposis coli (APC) 

protein and plus end binding proteins stabilize MTs by increasing the duration of the MT elongation phase. MT instability is promoted by several nonmotile kinesins from the 

kinesin-13 family. The mitotic centromere associated kinesin, MCAK, one of the most studied kinesin-13 family proteins, binds both plus and minus MT ends in vitro. The 

binding of MCAK to a MT end is thought to accelerate the transition to catastrophe by weakening the lateral interactions between the protofilaments. 

Tubulin undergoes several post-translational modifications such as acetylation, poly-glutamylation, and poly-glycylation, which have been shown to alter the association with 

certain MT motors as well as other proteins that can affect MT stability and dynamics. 


Anitei, M. and Hoflack, B. (2011) Nat. Cell Biol. 14, 11–19. • de Forges, H., Bouissou, A., and Perez, F. (2012) Int. J. Biochem. Cell Biol. 44, 266–274. • Etienne-Manneville, 

S. (2010) Curr. Opin. Cell Biol. 22, 104–111. • Mimori-Kiyosue, Y. (2011) Cytoskeleton (Hoboken) 68, 603–618. • van der Vaart, B., Akhmanova, A., and Straube, A. 

(2009) Biochem. Soc. Trans. 37, 1007–1013. 


Types of Endosomes 

Endosomes are membrane-bound vesicles that transport molecules from the plasma membrane to 

the lysosomes for degradation and can be categorized as early, late, or recycling endosomes. Early 

endosomes fuse with clathrin-coated endocytic vesicles that contain extracellular particles, fluid, and 

membrane-bound receptors. EEA1 is an early endosome marker essential for membrane fusion and 

trafficking. Early endosomes increase in size and undergo a maturation process that results in their development 

into late endosomes, which can be identified using late endosome markers Rab7 and Rab9. 

Late endosomes fuse with lysosomes, which results in degradation of vesicle contents. Molecules contained 

within early endosomes can also be transported to the Golgi for sorting via recycling endosomes. 

EEA1 is a marker for early endosomes. 

EEA1 (C45B10) Rabbit mAb #3288: Confocal IF analysis of HeLa cells 

using #3288 (green). Actin filaments have been labeled with DY-554 

phalloidin (red). Blue pseudocolor = DRAQ5 ® (fluorescent DNA dye). 

© 2008–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. James Bamburg, Colorado State University, Fort Collins, CO for reviewing this diagram. 


www.cellsignal.com/cstproteinfolding 117


Rab7 localizes to 

late endosomes. 

Rab7 (D95F2) XP ® Rabbit mAb 

#9367: Confocal IF analysis of SK- 

MEL-28 cells using #9367 (green). Actin 

filaments were labeled using DY-554 



LAMP1 localizes 

to lysosomes. 

LAMP1 (D2D11) XP ® Rabbit mAb 

#9091: Confocal IF analysis of HeLa 





Rab Proteins 

Rab proteins have been implicated in the regulation of intracellular protein trafficking in both the 

endocytic and biosynthetic pathways. GDP-bound Rabs are inactive and localize to the cytosol, while 

active GTP-bound Rabs localize to the cytoplasmic side of membrane compartments where they recruit 

downstream effector proteins that, along with Rabs, mediate vesicle formation, motility, docking, and 

fusion. Rab effector proteins include coat proteins, sorting adaptors, SNAREs, and microtubule- and 

actin-based motor proteins. 

The Rab11 subfamily (Rab11a, Rab11b, and Rab25) localizes to the endosomal recycling compartment, 

as well as to the apical recycling endosomes of polarized epithelial cells. Endosomal trafficking events 

through these compartments are mediated by Rab11 family members and their effector molecules, such 

as the Rab11-family interacting proteins (FIPs), Rabphilin-11/Rab11BP, myosin Vb, and Sec15. 

Rab proteins 

serve as markers 

for the various types 

of endosomes. 

Compartment 

Early Endosome 

Late Endosome 

Lysosome 

Recycling Endosome 

Perinuclear and Apical Recycling Endosome 

Golgi-to-Membrane Anterograde Endosome 

Exocytosis 

Endoplasmic Reticulum 

Golgi Apparatus 

ER-to-Golgi Anterograde Endosome 

Golgi-to-ER Retrograde Endosome 

Trans Golgi Network-to-Basolateral Membrane Endosome 

Rab25, which associates with apical recycling 

vesicles, is expressed in multiple cell lines. 

A 

Rab10, an early endosome marker, mediates 

protein transport between early endosomes 

and basolateral compartments. 

Rab10 (D36C4) XP ® Rabbit mAb #8127: Confocal IF analysis of MCF7 cells 

using #8127 (green). Actin filaments were labeled with DY-554 phalloidin (red). 

Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). 

Marker 

EEA1, Rab4, Rab5, Rab10 

Rab7, Rab9 

LAMP1 

Rab4, Rab11, Rab25, Rab35 

Rab17 

Rab38 

Rab3A 

PDI, Calnexin 

RCAS1 

Rab1A 

Rab6 

Rab8 

Rab25 (D4P6P) XP ® Rabbit mAb #13048: Confocal IF analysis of MCF7 (positive) (A) and HeLa (negative) (B) cells using #13048 

(green). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). WB analysis of extracts from various cell lines (C) using #13048. 


Barr, F.A. (2013) J. Cell Biol. 202, 191−199. • Gurgis, F.M., Ziaziaris, W., and Munoz, L. (2014) Mol. Pharmacol. 85, 345−356. 

• Lachance, V., Angers, S., and Parent, J.L. (2014) Small GTPases 5, e29039. • Mayer, M.P. (2013) Trends Biochem. Sci. 38, 

507−514. • Mendoza, P., Diaz, J., Silva, P., et al. (2014) Small GTPases. 5, e28195 • Pfeffer, S.R. (2013) Curr. Opin. Cell 

Biol. 25, 414−419. • Saibil, H. (2013) Nat. Rev. Mol. Cell Biol. 14, 630−642. • Villarroel-Campos, D., Gastaldi, L., Conde, C., 

et al. (2014) J. Neurochem. 129, 240−248. 


B 

C 

kDa 

80 

60 

50 

40 

30 

20 

10 

1 2 3 

Lanes 

1. HCT-15 

2. ZR-75-1 

3. MCF7 

Rab25 

Commonly Studied Protein Folding and Vesicle Trafficking Targets 

Target M P Target M P 

ACE2 

• Grp75 

• • 

APPL1 

• Grp94 

• 

Arf6 

• • Hip 

• • 

Bag1 

• Hop 

• • 

BAG6 

• HRS 

• 

BiP 

• • HSF1 

• • 

Calnexin 

• • HSP27 

• • 

Calpastatin 

• Phospho-HSP27 (Ser15) • 

Caveolin-1 • • Phospho-HSP27 (Ser78) • 

Phospho-Caveolin-1 (Tyr14) • Phospho-HSP27 (Ser82) • • 

CCT2 

• HSP40 

• • 

CDC37 

• • HSP60 

• • 

Phospho-CDC37 (Ser13) • HSP70 

• • 

CHOP 

• TRAP1/HSP75 • 

Clathrin Heavy Chain • • HSP90 

• • 

CRYAB 

• Phospho-HSP90α (Thr5/7) • 

Derlin-1 

• HSP90β 

• • 

DNAJC2/MPP11 • HSPA4/Apg-2 • 

Phospho-DNAJC2/MPP11 • HSPA8 

• 

(Ser47) 

HSPB8/HSP22 • 

Dynamin-I • 

HYOU1 

• 

Dynamin I/II 

• 

IGF-II Receptor/CI-M6PR • 

EEA1 

• • 

IRE1α 

• 

Eps15 

• • 

MBTPS2 

• 

Ero1-Lα 

• 

NSF 

• • 

ERp44 

• • 

OCRL1 

• 

ERp57 

• 

OS-9 

• 

ERp72 

• • 

p58IPK 

• 

FKBP4 

• • 

PDI 

• • 

GCN2 

• 

PERK 

• 

GM130 

• • 

Phospho-PERK (Thr980) • 

GOPC 

• 


Kalwa, H. et al. (2014) Central role 

for hydrogen peroxide in P2Y1 ADP 

receptor-mediated cellular responses 

in vascular endothelium. Proc. Natl. 

Acad. Sci. USA 111, 3383−3388. 

Gai, X. et al. (2014) Caveolin-1 is 

up-regulated by GLI1 and contributes 

to GLI1-driven EMT in hepatocellular 

carcinoma. PLoS One 9, e84551. 

Cai, B. et al. (2014) Rapid degradation 

of the complement regulator, 

CD59, by a novel inhibitor. J. Biol. 

Chem. 289, 12109−12125. 

Yi, S.L. et al. (2014) Role of 

caveolin-1 in atrial fibrillation as an 

anti-fibrotic signaling molecule in 

human atrial fibroblasts. PLoS One 

9, e85144. 

Huang, J. et al. (2014) Cross-talk between 

EphA2 and BRaf/CRaf is a key 

determinant of response to Dasatinib. 


Mukherjee, R. et al. (2014) Sexdependent 

expression of caveolin 1 

in response to sex steroid hormones 

is closely associated with development 

of obesity in rats. PLoS One 

9, e90918. 

Meckes, D.G. Jr. et al. (2013) 

Epstein-Barr virus LMP1 modulates 

lipid raft microdomains and the 

vimentin cytoskeleton for signal 

transduction and transformation. 

J. Virol. 87, 1301−1311. 

Randazzo, D. et al. (2013) Obscurin is 

required for ankyrinB-dependent dystrophin 

localization and sarcolemma 

integrity. J. Cell Biol. 200, 523−536. 

Choi, C.H. et al. (2013) Mechanism 

for the endocytosis of spherical 

nucleic acid nanoparticle conjugates. 

Proc. Natl. Acad. Sci. USA 110, 

7625−7630. 

Lin, H.Y. et al. (2013) Caveolar 

endocytosis is required for human 

PSGL-1-mediated enterovirus 71 

infection. J. Virol. 87, 9064−9076. 

Pongrakhananon, V. et al. (2013) 

Ouabain suppresses the migratory 

behavior of lung cancer cells. PLoS 

One 8, e68623. 

Knowles, C.J. et al. (2013) Palmitate 

diet-induced loss of cardiac 

caveolin-3: a novel mechanism for 

lipid-induced contractile dysfunction. 


Target M P 

PKR 

Prostate Specific Membrane 

Antigen 

Rab1A 

Rab3A 

Rab4 

Rab5 

Rab6 

Rab7 

Rab8 

Rab9 

Rab10 

Rab11 

Rab11a 

Rab11b 

Rab11FIP1 

Rab17 

Rab25 

Rab35 

Rab38 

Rabex-5 

RBX1 

RCAS1 

REPS1 

SGTA 

Phospho-SGTA (Ser305) 

SNIP/p140Cap 

STAM1 

Syntaxin 6 

Tid-1 

VAMP8 

• • 

• 

• 

• • 

• 

• • 

• • 

• • 

• 

• 

• • 

• • 

• 

• 

• 

• 

• • 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• 

• 

• 

• 

Nho, R.S. et al. (2013) FoxO3a 

(Forkhead Box O3a) deficiency 

protects Idiopathic Pulmonary 

Fibrosis (IPF) fibroblasts from type I 

polymerized collagen matrix-induced 

apoptosis via caveolin-1 (cav-1) and 

Fas. PLoS One 8, e61017. 

Park, W.J. et al. (2013) Protection of a 

ceramide synthase 2 null mouse from 

drug-induced liver injury: role of gap 

junction dysfunction and connexin 32 

mislocalization. J. Biol. Chem. 288, 

30904−30916. 

Demir, K. et al. (2013) RAB8B is 

required for activity and caveolar 

endocytosis of LRP6. Cell Rep. 4, 

1224−1234. 

Simone, L.C. et al. (2013) Role of 

phosphatidylinositol 4,5-bisphosphate 

in regulating EHD2 plasma membrane 

localization. PLoS One 8, e74519. 

Ekman, M. et al. (2013) Mir-29 

repression in bladder outlet obstruction 

contributes to matrix remodeling 

and altered stiffness. PLoS One 8, 

e82308. 


These protein targets are commonly 

studied in protein folding and vesicle 

trafficking research. Primary antibodies, 








32 


CST antibodies for Caveolin 




www.cellsignal.com/cstproteinfolding 119



Ubiquitin and Ubiquitin-like Proteins 

The ubiquitin-proteasome system (UPS) is the primary means by which cellular proteins are degraded. 

The UPS is a highly regulated system for elimination of misfolded or damaged proteins as well as 

proteins whose activity is acutely regulated by signaling pathways. This system plays a central role 

in cell proliferation, transcriptional regulation, apoptosis, immunity, development, and many other 

cellular processes (e.g., organelle biogenesis, cellular response to infection, etc.). Ubiquitin is a highly 

conserved 76-amino acid protein that can be covalently linked to many cellular proteins through an enzymatic 

cascade. Ubiquitination is an ATP-dependent process carried out by three classes of enzymes. 

A “ubiquitin activating enzyme” (E1), UBA1, forms a thio-ester bond with ubiquitin. This reaction allows 

subsequent binding of ubiquitin to “ubiquitin conjugating enzymes” (E2s), followed by the formation of 

an isopeptide bond between the C-terminus of ubiquitin and the ε-amino group of a lysine residue on 

the substrate protein. The latter reaction requires a “ubiquitin ligase” (E3). Several hundred E3 ligases 

exist within the eukaryotic cell; each ligase can only modify a subset of substrate proteins, thereby 

providing substrate specificity to the system. Ubiquitinated proteins are then targeted to the 26S 

proteasome for degradation or experience changes in protein location or activity. 

Ubiquitin Linkages 

Ubiquitin can be linked to a substrate as a single unit, monoubiquitination, or as a branched chain, 

polyubiquitination. Substrate proteins are linked to ubiquitin using seven distinct ubiquitin lysine residues 

(Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63). Formation of a polyubiquitin chain occurs 

when a lysine residue of ubiquitin is linked to the C-terminal glycine of another ubiquitin. Polyubiquitinated 

proteins have distinct fates depending upon the nature of the ubiquitin linkage through which 

they are conjugated; K48-linked polyubiquitin chains mainly target proteins for proteasomal degradation 

while K63-linked polyubiquitin chains typically regulate protein function, subcellular localization, 

and protein-protein interactions. The K63 linkage sometimes results in proteasomal degradation as 

well. K11-linked polyubiquitin chains regulate cell cycle targets and progression through mitosis.In 

addition, ubiquitin also can be linked to a target protein through its N-terminal methionine residue. 

This linkage, termed linear ubiquitination, is catalyzed by the linear ubiquitin chain assembly complex 

(LUBAC) and plays a critical role in NF-κB signaling. 

Distinct ubiquitin linkages can be detected using linkage-specific antibodies. 


Ub 

ATP 

E2 

E3 

Substrate 

E1 

E2 

Substrate 

Ub 

Ub 

E3 

Ub 

Polyubiquitination 

via multiple cycles 

Proteasomal Degradation 

Changes in Protein Location or Activity 

K63-linkage Specific 

Polyubiquitin (D7A11) 

Rabbit mAb #5621: WB 

analysis of seven distinct 

recombinant polyubiquitin 

chains (A) using #5621 

(upper) and Ubiquitin 

Antibody #3933 (lower). 


Polyubiquitin (D9D5) 

Rabbit mAb #8081: WB 

analysis of six distinct 

recombinant polyubiquitin 

chains (B) using #8081 

(upper) and Ubiquitin (P4D1) 

Mouse mAb #3936 (lower). 

Lanes 

1. K6 

2. K11 

3. K27 

4. K29 

5. K33 

6. K48 

7. K63 

A 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

20 

200 

140 

100 

80 

60 

50 

40 

30 

20 

1 2 3 4 5 6 7 

K63-linked 

Polyubiquitin 

Polyubiquitin 

B 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

20 

200 

140 

100 

80 

60 

50 

40 

30 

20 

1 2 4 5 6 7 

K48-linked 

Polyubiquitin 

Polyubiquitin 

UBE3A, a widely expressed E3 ubiquitin ligase 

UBE3A (D10D3) Rabbit mAb #7526: WB analysis 


Lanes 

1. K-562 

2. SK-N-SH 

3. SK-N-MC 

4. A172 

5. T-47D 

6. HEK001 

7. T24 

8. KNRK 

9. NIH/3T3 

10. COS-7 

kDa 

200 

140 

100 

80 

60 

1 2 3 4 5 6 7 8 9 10 

UBE3A 

Deubiquitinating Enzymes 

Deubiquitinating enzymes (DUBs) reverse the process of ubiquitination by removing ubiquitin from its 

substrate protein. DUB activity maintains ubiquitin recycling and ensures the cellular pool of ubiquitin 

molecules remains steady. DUBs are categorized into 5 subfamilies: USP, UCH, OTU, MJD, and JAMM, 

each with a specific tissue and ubiquitin-linkage specificity. Overexpression or misregulation of DUBs 

have been linked to cancer and other diseases. For example, overexpression of USP6 has been linked 

to bone and other mesenchymal tumors, mutations in CYLD are associated with skin tumors, and 

overexpression of USP33 to von Hippel-Lindau diease. In addition, other DUB mutations are associated 

with neurodegenerative disorders, including a role for UCHL1 in Parkinson’s disease and ATX3 in 

spinocerebellar ataxia. 

Skp2, a substrate 

recognition subunit of 

the Skp-Cullin-F-box 

(SCF) ubiquitin ligase 

complex, is expressed 

in many cell lines 

and cancers. 

A 

B 

kDa 

100 

80 

60 

50 

40 

30 

1 2 3 4 5 6 7 8 9 10 

Skp2 (D3G5) XP ® Rabbit mAb #2652: IHC analysis of paraffin-embedded human ovarian carcinoma (A) using #2652. 

WB analysis of extracts from various cell lines (B) using #2652. 

Skp2 

Lanes 

1. U-2 OS 

2. LNCaP 

3. MDA- 

MB-468 

4. K-562 

5. U-87 MG 

6. WI-3B 

7. SK-OV-3 

8. MCF7 

9. MCF-10A 

10. COS-7 

A 

B 

kDa 1 2 3 4 5 6 7 8 9 10 

200 

140 

100 

80 

60 

USP10 (D7A5) Rabbit mAb #8501: Confocal IF analysis of HCT 116 cells (A) using #8501 (green). Blue pseudocolor 

= DRAQ5 ® #4084 (fluorescent DNA dye). WB analysis of extracts from various cell lines (B) using #8501. 

USP10 

Lanes 

1. HCT 116 

2. NCI-H23 

3. 786-0 

4. U-2 OS 

5. LNCaP 

6. MCF7 

7. Meg-01 

8. mIMCD-3 

9. H-4-11-E 

10. COS-7 

USP10, a DUB known 

to act on p53, Vps34, 

and CFTR, is found 

in the cytoplasm and 

is widely expressed in 



www.cellsignal.com/cstubiquitin 

121



UCHL1 (D3T2E) XP ® Rabbit 

mAb #13179: WB analysis of 

extracts from various cell lines (A) 

using #13179 (upper) and GAPDH 

(D16H11) XP ® Rabbit mAb #5174 

(lower). IHC analysis of paraffinembedded 

human non-small cell 

lung carcinoma (B) using #13179. 

Lanes 

1. DU 145 

2. LNCaP 

3. NCI-H1299 

4. NCI-H358 

5. WI-38 

6. BT-549 

7. U266 

8. A172 

9. SH-SY5Y 

10. T98G 

11. SK-N-AS 

12. Neuro-2a 

13. C6 

14. COS-7 

SENP3, a SUMO 

protease, localizes 

to the nucleolus. 

SENP3 (D20A10) XP ® Rabbit mAb 

#5591: Confocal IF analysis of HeLa cells 

using #5591 (green). Actin filaments 




Interferon-γ 

induces expression 

of PSMB8, a core 

particle subunit of the 

immunoproteasome. 

kDa 

140 

100 

80 

60 

50 

40 

30 

20 

60 

50 

40 

30 

1 

– + 

– + 

PSMB8/LMP7 

(precursor) 

PSMB8/LMP7 

(mature) 

GAPDH 

hIFN-γ 

PSMB8/LMP7 (1A5) Mouse mAb 


HeLa and SW620 cells, untreated or 

treated with Human Interferon-γ (hIFN-γ) 

#8901 (100 ng/ml, 72 hr), using 

#13726 (upper) and GAPDH (D16H11) 

XP ® Rabbit mAb #5174 (lower). 

2 

Lanes 

1. HeLa 

2. SW620 

UCHL1 is expressed in many cell lines 

and human non-small cell lung carcinoma. 

A 

kDa 

80 

60 

50 

40 

30 

20 

10 

50 

40 

30 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Sumoylation and Neddylation 

Small ubiquitin-related modifier 1, 2, and 3 (SUMO-1, -2, and -3) and NEDD8 are members of the 

ubiquitin-like protein family. SUMO and NEDD8 can be covalently attached to proteins (termed 

sumoylation and neddylation, respectively) in a manner analogous to ubiquitination using an E1, E2, E3 

conjugation system. Unlike ubiquitination, however, sumoylation and neddylation of substrate proteins 

does not typically result in degradation. Instead, the SUMO and NEDD modifications affect subcellular 

localization, protein function, or protein-protein interactions. 

Sumoylation substrates include RanGAP, PML, p53, IκB-α, topoisomerase II, and APP. Sumoylation has 

numerous cellular effects including nuclear trafficking, regulation of transcriptional activity, and protein 

stability. SUMO modifications can be removed (desumoylation) by proteases such as SENP3, which 

catalyzes the release of SUMO2 and SUMO3 monomers from sumoylated substrates. 

Neddylation is the covalent attachment of NEDD8 via its C-terminal glycine residue to a lysine residue 

within the target protein. In contrast to the ubiquitin pathway, only a handful of neddylation substrates 

are known to date. NEDD8 and ubiquitin modifications are closely connected in that neddylation is an 

important regulator of E3 ubiquitin ligase activity. For example, neddylation of cullin proteins activates 

the SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase complex by promoting complex formation and enhancing 

the recruitment of the E2-ubiquitin intermediate. Other neddylation substrates include p53, Mdm2, 

RPL11, and VHL. 

Proteasome 

The 26S proteasome is a highly abundant proteolytic complex involved in the degradation of ubiquitinated 

substrate proteins. It consists largely of two sub-complexes, the 20S catalytic core particle (CP) 

and the 19S/PA700 regulatory particle (RP) that can cap either end of the CP. The CP consists of two 

stacked heteroheptameric β-rings (β1-7) that contain three catalytic β-subunits and are flanked on either 

side by two heteroheptameric α-rings (α1-7). The RP includes a base and a lid, each having multiple 

subunits. The base, in part, is composed of a heterohexameric ring of ATPase subunits belonging 

to the AAA (ATPases Associated with diverse cellular Activities) family. The ATPase subunits function to 

unfold the substrate and open the gate formed by the α-subunits, thus exposing the unfolded substrate 

to the catalytic β-subunits. The lid consists of ubiquitin receptors and DUBs that function in recruitment 

of ubiquitinated substrates and modification of ubiquitin chain topology. Other modulators of 

proteasome activity, such as PA28/11S REG, can also bind to the end of the 20S CP and activate it. 

Proteasome activity can be inhibited by the chemical modulator bortezomib. 

Constitutively expressed core particle subunits PSMB5, PSMB7, and PSMB6 provide chymotrypsin-like, 

trypsin-like, and caspase-like activities, respectively. In immune cells involved in antigen presentation, 

these subunits are replaced by highly homologous, induced β-subunits PSMB8, PSMB9, and PSMB10 

to form the immunoproteasome. The immunoproteasome functions in degradation of proteins into fragments 

of the correct size for presentation on MHC class I molecules. 

UCHL1 

GAPDH 

B 


Amm, I., Sommer, T., and Wolf, D.H. (2014) Biochim. Biophys. Acta. 1843, 182−196. • Bhattacharyya, S., Yu, H., and Mim, 

C. (2014) Nat. Rev. Mol. Cell Biol. 15, 122−133. • Rabut, G. and Peter, M. (2008) EMBO Rep. 9, 969−976. • Raule, M., 

Cerruti, F., and Cascio, P. (2014) Biochim. Biophys. Acta. 1843, 1942−1947. • Rieser, E., Cordier, S.M., and Walczak, H. 

(2013) Trends Biochem. Sci. 38, 94−102. • Ruggiano, A., Foresti, O., and Carvalho, P. (2014) J. Cell Biol. 204, 869−879. 

• Schulman, B.A. and Harper, J.W. (2009) Nat. Rev. Mol. Cell Biol. 10, 319−331. • Singhal, S., Taylor, M.C., and Baker, R.T. 

(2008) BMC Biochem. 9, S3. • Sriramachandran, A.M. and Dohmen, R.J. (2014) Biochim. Biophys. Acta. 1843, 75−85. • 

Zhao, Y., Brickner, J.R., and Majid, M.C. (2014) Trends Cell Biol. 24, 426−434. 

Commonly Studied Ubiquitin Targets 

Target M P S 

ADRM1 

• • • 

AMFR 

• 

APC1 

• 

APC2 

• • 

APC3 

• • • 

APC11 

• • 

BAP1 

• 

Phospho-BAP1 (Ser592) 

• 

β-Trcp 

• 

CAND1 

• • • 

c-Cbl 

• • 

Phospho-c-Cbl (Tyr700) 

• 


• 


• 

Cbl-b 

• • • 

UBC3 

• 

CHIP 

• 

COPS5 

• • 

CUL1 

• 

CUL3 

• 

CUL4A 

• 

CYLD 

• • 

Phospho-CYLD (Ser418) 

• 

DDB-1 

• • 

DDB-2 

• 

E2-25K/Hip2 

• • 

HAUSP 

• • 

HECTH9 

• 

ISG15 

• • • 

ITCH 

• 

KEAP1 

• • • 

KLHL12 

• • 

MIB1 

• 

NAE1/APPBP1 

• 

NEDD4 

• • 

NEDD4L 

• 

• 

Phospho-NEDD4L 

(Ser342) 

Target M P S 

Phospho-NEDD4L 

(Ser448) • 

NEDD8 

• • 

NPL4 

• 

OTUB1 

• 

OTULIN 

• 

PA28α 

• • 

PA28β 

• 

PA28γ 

• 

PSMA2 

• • 

PSMA3 

• • 

PSMA5 

• 

PSMA6 

• 

PSMB5 

• • 

PSMB6 

• 

PSMB7 

• • 

PSMB8/LMP7 

• 

PSMC3/TBP1 

• 

PSMC5/TRIP1 

• 

PSMD10/Gankyrin 

• 

PSMD11 

• 

PSMD14 

• • 

Rad23B 

• 

RBX1 

• • 

RCHY1 

• 

S5a/PSMD4 

• • 

SENP1 

• 

SENP3 

• 

Sharpin 

• 

Skp1 

• • • 

Skp2 

• • • 

SPINK3 

• 

STAMBP 

• 

SUMO-1 

• • 

SUMO-2/3 

• 

SYVN1 

• • 

TRIAD1 

• • 

TRIM25 

• 

Target M P S 

TRIM27 

• 

UBA2 

• • • 

UBC3B 

• 

Ubc9 

• • 

Ubc12 

• • 

Ubc13 

• 

UbcH5C 

• 

UBE1a 

• 

UBE1a/b 

• 

UBE1L2/UBA6 

• 

UBE2C 

• 

UBE2L3 

• • 

UBE2N 

• 

UBE2S 

• • 

UBE2T 

• • 

UBE3A 

• 

Ubiquitin 

• • 

• • 


Polyubiquitin 


Polyubiquitin 

UBLE1A/SAE1 

• 

• 

UBR5 

• 

UCHL1 

• • • 

UCHL3 

• • • 

USP1 

• • 

USP2 

• 

USP4 

• 

USP8 

• • 

USP9X 

• • 

USP10 

• • • 

USP13 

• 

USP14 

• • • 

USP18 

• 

VCP 

• • 

VHL 

• 

VPRBP 

• 


nodes within ubiquitin signaling 

pathways and are commonly studied in 

ubiquitin research. Primary antibodies, 









7 


CST antibodies for NEDD4 have been 

cited over 7 times in high-impact, peerreviewed 




Sun, Y. et al. (2014) Histone deacetylase 

5 blocks neuroblastoma cell differentiation 

by interacting with N-Myc. 

Oncogene 33, 2987−2994. 

Liu, P.Y. et al. (2013) The histone 

deacetylase SIRT2 stabilizes Myc 

oncoproteins. Cell Death Differ. 20, 

503−514. 

Adler, J.J. et al. (2013) Amot130 

adapts atrophin-1 interacting protein 

4 to inhibit yes-associated protein 

signaling and cell growth. J. Biol. 

Chem. 288, 15181−15193. 

Xia, P. et al. (2013) WASH inhibits 

autophagy through suppression of 

Beclin 1 ubiquitination. EMBO J. 32, 

2685−2696. 

Fu, J. et al. (2013) The short isoform 

of the ubiquitin ligase NEDD4L is a 

CREB target gene in hepatocytes. 


Andersen, M.N. et al. (2013) A 

phosphoinositide 3-kinase (PI3K)- 

serum- and glucocorticoid-inducible 

kinase 1 (SGK1) pathway promotes 

Kv7.1 channel surface expression by 

inhibiting Nedd4-2 protein. J. Biol. 

Chem. 288, 36841−36854. 

Liao, C.K. et al. (2013) Lipopolysaccharide 

induces degradation of 

connexin43 in rat astrocytes via the 

ubiquitin-proteasome proteolytic 

pathway. PLoS One 8, e79350. 


www.cellsignal.com/cstubiquitin 123



Ubiquitin/Proteasome 

E1 

Ub 

DUBs 

Ub 

Ub 

Ub 

ATP 

AMP 

Ub Ub 

Ub 

Ub 

ATP 

ADP 

E1 

Ub Ub 

Ub 

Ub 

Ub 

Peptides 

Ub Ub 

Ub 

Ub 

E2 

DUBs 

E1 

Ub Ub 

Ub 

Ub 

E2 

Ub 

Ub Ub 

Ub 

Ub 

26S 

Proteasome 

Protein 

Substrate 

E3 

Ub Ub 

Ub 

Ub 

E2 

E3 

Ub 

Ub Ub 

Ub 

Ub 

Ub 

Mono-Ub 

Multiple 

Cycles 

Ub 

Ub 

Multi-Mono-Ub 

Endocytosis 

DNA Repair 

Protein Localization 

Trafficking 

Ub Ub 

Ub 

Ub 

Ub Ub 

Ub 

Ub 

K48 Poly-Ub K11 Poly-Ub K6 Poly-Ub K27 Poly-Ub K29 Poly-Ub K33 Poly-Ub K63 Poly-Ub Linear Poly-Ub 

19S 

20S 

19S 

DNA Damage 

Response 

TCR Signaling 

Lysomal 

Degradation 

Mitochondrial 

Maintenance and 

Mitophagy 

Activating NF-κB 

Signaling 

The ubiquitin proteasome pathway, conserved from yeast to mammals, is required for the targeted degradation of most shortlived 

proteins in the eukaryotic cell. Targets include cell cycle regulatory proteins, whose timely destruction is vital for controlled 

cell division, as well as proteins unable to fold properly within the endoplasmic reticulum. 

Ubiquitin modification is an ATP-dependent process carried out by three classes of enzymes. A “ubiquitin activating enzyme” (E1) 

forms a thio-ester bond with ubiquitin, a highly conserved 76-amino acid protein. This reaction allows subsequent binding of 

ubiquitin to a “ubiquitin conjugating enzyme” (E2), followed by the formation of an isopeptide bond between the carboxy-terminus 

of ubiquitin and a lysine residue on the substrate protein. The latter reaction requires a “ubiquitin ligase” (E3). E3 ligases can 

be single- or multi-subunit enzymes. In some cases, the ubiquitin-binding and substrate binding domains reside on separate 

polypeptides brought together by adaptor proteins or cullins. Numerous E3 ligases provide specificity in that each can modify only 

a subset of substrate proteins. Further specificity is achieved by post-translational modification of substrate proteins, including, 

but not limited to, phosphorylation. 

Effects of monoubiquitination include a role in endocytosis and DNA damage, as well as changes in subcellular protein localization 

and trafficking. However, multiple ubiquitination cycles resulting in a polyubiquitin chain are required for targeting a protein 

to the proteasome for degradation. The multisubunit 26S proteasome recognizes, unfolds, and degrades polyubiquitinated 

substrates into small peptides. The reaction occurs within the cylindrical core of the proteasome complex, and peptide bond 

hydrolysis employs a core threonine residue as the catalytic nucleophile. Polyubiquitin chains are also indicated in diverse cellular 

processes including DNA damage response, mitochondrial maintenance and mitophagy, lysosomal degradation, T Cell Receptor 

signaling, and NF-κB signaling. 

Ubiquitinating enzymes (UBEs) catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) 

action. Five DUB subfamilies are recognized, including the USP, UCH, OTU, MJD, and JAMM enzymes. In humans, there are three 

proteasomal DUBs: PSMD14 (POH1/RPN11), UCH37 (UCH-L5), and Ubiquitin-Specific Protease 14, which is also known as the 

60 kDa subunit of tRNA-guanine transglycosylase (USP14/TGT60 kDa). 


Amm, I., Sommer, T., and Wolf, D.H. (2014) Biochim. Biophys. Acta. 1843, 182–196. • Budhidarmo, R., Nakatani, Y., and 

Day, C.L. (2012) Trends Biochem. Sci. 37, 58–65. • Burrows, J.F. and Johnston, J.A. (2012) Front. Biosci. 17, 1184–1200. 

• Campello, S., Strappazzon, F., and Cecconi, F. (2014) Biochim. Biophys. Acta. 1837, 451–460. • Corn, J.E. and Vucic, D. 

(2014) Nat. Struc. Mol. Biol. 21, 297–300. • Hammond-Martel, I., Yu, H., Affar, el B. (2012) Cell Signal. 24, 410–421. • Hildebrand, 

J.M., Yi, Z., Buchta, C.M., Poovassery, J., Stunz, L.L., and Bishop, G.A. (2011) Immunol. Rev. 244, 55–74. • Hurley, 

J.H. and Schulman, B.A. (2014) Cell 157, 300–311. • Ruggiano, A., Foresti, O., and Carvalho, P. (2014) J. Cell Biol. 204, 

869–879. • Schaefer, A., Nethe, M., and Hordijk, P.L. (2012) Biochem. J. 442, 13–25. • Tokunaga, F. (2013) J. Biochem. 

154, 313–323. • Weissman, A.M., Shabek, N., and Ciechanover, A. (2011) Nat. Rev. Mol. Cell Biol. 12, 605–620. • Zhao, Y., 

Brickner, J.R., Majid, M.C., and Mosammaparast, N. (2014) Trends Cell Biol. 24, 426–434. 

Ligase Substrate Function PMID 

AMFR KAI1 AMFR is also known as gp78. AMFR is an integral ER membrane protein and 

functions in ER-associated degradation (ERAD). AMFR has been found to 

promote tumor metastasis through ubiquitination of the metastasis suppressor, 

KAI1. 

18037895 

APC/CDC20 Cyclin B, 

Securin 

APC/Cdh1 

CDC20, Cyclin 

B, Cyclin A, 

Aurora A, Skp2, 

Claspin 

The anaphase promoting complex/cyclosome (APC/C) is a multiprotein 

complex with E3 ligase activity that regulates cell cycle progression through 

degradation of cyclins and other mitotic proteins. APC is found in a complex 

with CDC20, CDC27, SPATC1, and TUBG1. 

The anaphase promoting complex/cyclosome (APC/C) is a multiprotein 

complex with E3 ligase activity that regulates cell cycle progression through 

degradation of cyclins and other mitotic proteins. The APC/C-Cdh1 dimeric 

complex is activated during anaphase and telophase, and remains active until 

onset of the next S phase. 

ARIH1 4EHP ARIH1 is an E3 ubiquitin ligase that may regulate protein translation by targeting 

eIF4E2 for ubiquitination and degradation by the proteasome. 

BIRC2 Smac, TRAF2 BIRC2 is an apoptotic suppressor that prevents caspase activation by forming 

a complex with TNF receptor associated factors 1 and 2 (TRAF1 and TRAF2), 

which is then recruited to the tumor necrosis factor receptor 2 (TNFR2). 

BIRC3 Caspase 3 

and 7, Smac, 

TRAF1 

BIRC4 Caspase 3, 

Smac, MEKK2 

BIRC3 is an apoptotic suppressor that prevents caspase activation by forming 


which is then recruited to the tumor necrosis factor receptor 2 (TNFR2). 

BIRC4 is an apoptotic suppressor that prevents caspase activation by forming 


which is then recruited to the tumor necrosis factor receptor 2 (TNFR2). BIRC4 

is also known as XIAP. 

BIRC7 Smac BIRC7 is an E3 ubiquitin ligase with anti-apoptotic activity. BIRC7 supports 

cell survival by targeting Smac for ubiquitination and degradation by the 

proteasome. 

Bmi1 H2A K119 Bmi1 is a component of the polycomb group multiprotein PRC1- like (PcG 

PRC1) complex. Bmi1 is required for stimulating PcG PRC1 ubiquitin-protein 

ligase activity. 

BRCA1 

ER-a, Rpb8, 

CtIP, FANCD2 

BRCA1 is an E3 ubiquitin ligase that maintains genomic stability by repairing 

DNA damage. Research studies have shown that mutations of this gene have 

been linked to breast cancer. 

C6orf157 Cyclin B C6orf157 is also known as H10BH. C6orf157 is an E3 ubiquitin ligase that has 

been shown to ubiquitinate cyclin B. 

Cbl 

Cbl-b and c-Cbl are members of the Cbl family of adaptor proteins that are 

highly expressed in hematopoietic cells. Cbl proteins possess E3 ubiquitin 

ligase activity that downregulates numerous signaling proteins and RTKs in several 

pathways such as EGFR, T cell and B cell receptors, and integrin receptors. 

Cbl proteins play an important role in T cell receptor signaling pathways. 

CBLL1 CDH1 CBLL1 is also known as Hakai. CBLL1 is an E3 ubiquitin ligase that ubiquitinates 

the phosphorylated form of E-Cadherin, causing its degradation and loss 

of cell-cell adhesions. 

CHFR PLK1, Aurora A CHFR is an E3 ubiquitin ligase that functions as a mitotic stress checkpoint 

protein that delays entry into mitosis in response to stress. CHFR has been 

shown to ubiquitinate and degrade the kinases PLK1 and Aurora A. 

CHIP 

CUL3/ 

BACURD 

CUL3/HIB/ 

SPOP 

HSP70/90, 

iNOS, Runx1, 

LRRK2 

RhoA 

Ci/Gli 

CHIP is an E3 ubiquitin ligase that acts as a co-chaperone protein and interacts 

with several heat shock proteins, including HSP70 and HSP90, as well as 

the nonheat shock proteins iNOS, Runx1, and LRRK2. 

CUL3/BACURD is a ubiquitin ligase complex composed of CUL3 and the BTB 

domain adaptor BACURD. CUL3/BACURD controls actin cytoskeleton structure 

and cell movement by promoting ubiquitination and degradation of small 

GTPase RhoA. 

CUL3/HIB/SPOP is an E3 ubiquitin ligase complex composed of Cullin3, 

Hedgehog-induced MATH and BTB domain-containing protein (HIB), and SPOP. 

CUL3/HIB/SPOP targets the Hedgehog pathway transcription factor (Ci)/Gli for 

ubiquitination and degradation by the proteasome. 

CUL3/KEAP1 Nrf2, IKKβ CUL3/KEAP1 is part of an E3 ubiquitin ligase complex composed of RBX1, 

CULlin3 and the substrate recognition component, KEAP1. CUL3/KEAP1 

targets Nrf2, a transcription factor that regulates antioxidant genes in response 

to oxidative stress for ubiquitination and degradation by the proteasome. In 

addition, CUL1/KEAP1 E3 ligase downregulates NF-κB signaling by targeting 

IKKβ ubiquitination. 

CUL3/ 

MEL-26 

mei-1 

CUL3/MEL-26 is an E3 ubiquitin ligase complex composed of Cullin3 and the 

substrate recognition component, MEL-26. MEL-26 targets mei-1 for ubiquitination 

and subsequent proteasomal degradation. 

17609108, 

12070128 

10548110, 

11562349, 

15014503, 

19477924 

14623119 

12525502, 

18434593 

10862606, 

12525502, 

15468071 

11447297, 

12121969, 

18761086 

16729033 

18650381 

17392432, 

17283126, 

16818604, 

11239454 

15749827 

18759930, 

9797470 

11836526 

14562038, 

19326084 

19913553, 

19362296, 

19524548, 

19536328 

19782033 

16740475 

12682069, 

19818716 

13679922 

Ubiquitin Ligases 

This table provides a list of E3 ubiquitin 

ligases, along with their substrates 

(when known), and corresponding 

references. This table was generated 

using PhosphoSitePlus ® , Cell Signaling 

Technology’s protein modification 

resource. 

© 2004–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Wenyi Wei, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA for reviewing this diagram. 






CUL3/Ctb9/ 

KLHDC5 

p60/katanin CUL3/Ctb9/KLHDC5 is an E3 ubiquitin ligase complex required for efficient 

p60/katanin removal through promoting ubiquitination of p60/katanin to allow 

normal mitotic progression in mammalian cells. 

19261606 

CUL3/KLHL3 WNK1, WNK4 CUL3/KLHL3 is an E3 ubiquitin ligase complex composed of Cullin3 and kelchlike 

3 (KLHL3) protein. WNK4 is a known target of CUL3/KLHL3-mediated 

ubiquitination and errors in that process are a common mechanism of human 

hereditary hypertension. Furthermore, mutations in KLHL3 and Cullin3 were 

identified to cause the human hypertensive disease pseudohypoaldosteronism 

type II (PHAII). 

CUL3/KLHL8 Rapsyn CUL3/KLHL8, an E3 ubiquitin ligase complex, controls rapsyn stability through 

polyubiquitination, which is required for clustering of nicotinic acetylcholine 

receptors (nAChRs) at the neuromuscular junction. 

CUL3/ 

KLHL12 

CUL3/ 

KLHL9/ 

KLHL13 

CUL3/ 

KLHL20 

CUL3/ 

KLHL22 

CUL3/ 

KLHL25 

CUL3/SPOP 

Dsh 

Aurora B 

PML, DAPK 

PLK1 

4E-BP1 

Gli2/Gli3, Daxx, 

SRC-3, AR, 

PTEN 

CUL4/CDT2 Cdt1, p21, 

Set8, CHK1 

CUL4/DDB1/ 

Cereblon 

IKZF1, IKZF3 

CUL4/COP1 c-Jun, p53, 

ETV1, ETV4, 

ETV5 

CUL3/KLHL12 E3 ubiquitin ligase targets Dishevelled for poly-ubiquitination 

and degradation, thus negatively regulating the Wnt/β-catenin pathway. 

CUL3/KLHL9/KLHL13 E3 ubiquitin ligase controls the dynamic behavior of 

mitotic chromosomes through ubiquitination of Aurora B, thereby coordinating 

mitotic progression and completion of cytokinesis. 

CUL3/KLHL20 E3 ubiquitin ligase mediates hypoxia-induced PML proteasomal 

degradation via activation by HIF-1. The CUL3/KLHL20 complex also controls 

interferon responses by promoting DAPK polyubiquitination and proteasomal 

degradation. 

CUL/KLHL22 regulates localization of PLK1 on the kinetochore thereby controlling 

spindle assembly checkpoint (SAC) activation. Ubiquitination of PLK1 

signals degradation-independent removal from kinetochores and fulfillment of 

SAC leading to mitotic progression. 

CUL3/KLHL25 E3 ubiquitin ligase complex targets hypophosphorylated 4E-BP1 

for degradation. Regulation of 4E-BP1 protein levels by CUL3/KLHL25 ubiquitination 

induces homeostatic control over the mRNA binding protein, eIF4E, for 

which 4E-BP1 acts as a repressor protein. 

The CUL3/SPOP E3 ubiquitin ligase displays both tumor suppressor and 

oncogenic functions in several types of human tissue. CUL3/SPOP regulates 

the proteolysis of the oncogene SRC-3, where underexpression of CUL3/ 

SPOP leads to overexpression of SRC-3 in prostate cancer. Inversely, SPOP, a 

direct transcriptional target of HIFs in some tissues, is overexpressed in 85% 

of kidney cancers. The opposing roles of SPOP protein in prostate and kidney 

cancers may result from degradation of different substrates, such as AR in 

prostate cancers and PTEN in kidney cancer. 

CUL4/CDT2 is an E3 ubiquitin ligase complex composed of DCX (DDB1-CUL4- 

X-box) and the substrate recognition component, CUL4/CDT2. CUL4/CDT2 

regulates cell cycle progression into S phase by targeting CDT1 and SPD1 for 


Cereblon forms an E3 ubiquitin ligase complex with CUL4 and DDB1 to regulate 

limb development. Lenalidomide-bound Cereblon targets IKZF1 and IKZF3 

for ubiquitination and degradation, helping to prevent B cell malignancies. 

CUL4/COP1 is an E3 ubiquitin ligase that mediates ubiquitination and subsequent 

proteasomal degradation of target proteins. COP1 targets the oncoprotein 

c-Jun, transcription factor ETV1 and may target the tumor suppressor p53 

for ubiquitination and degradation. 

CUL4/DDB2 XPC, H3, H4 CUL4/DDB2 is an E3 ubiquitin ligase complex composed of DCX (DDB1-CUL4- 

ROC1) and the substrate recognition component, CUL4/DDB2. CUL4/DDB2 

may target histone H2A, histone H3, and histone H4 at sites of UV-induced 

DNA damage to induce ubiquitination and degradation by the proteasome. 

CUL4/FBW5 TSC2 CUL4/FBW5 is an E3 ubiquitin ligase complex composed of DCX (DDB1-CUL4- 

ROC1) and the substrate recognition component, CUL4/FBW5. CUL4/FBW5 

regulates TSC2 protein stability and TSC complex turnover. 

CUL4/β-TrCP REDD1 CUL4/β-TrCP E3 ligase complex contains DCX (DDB1-CUL4-ROC1) and β-TrCP. 

This complex targets REDD1 for ubiquitination and subsequent proteasomal 

degradation to re-activate the mTOR signaling pathway as cells recover from 

hypoxic stress. 

CUL4/RBBP7 p150 CUL4/RBBP7 is an E3 ubiquitin ligase complex composed of DCX (DDB1- 

CUL4-ROC1) and the substrate recognition component, RBBP7. CUL4/RBBP7 

targets p150 for ubiquitination and degradation. 

CUL4/DDB1/ 

TRCP4A 

CUL4/VPRBP TET 

N-Myc, C-Myc 

CUL4/TRCP4A is an E3 ubiquitin ligase complex composed of DDB1-CUL4 and 

the substrate recognition component, TRCP4A. CUL4/DDB1/TRCP4A targets 

Myc for ubiquitination and degradation. 

CUL4/VPRBP is an E3 complex consisting of CRL4 (DDB1-CUL4-RBX1), and 

VPRBP (DCAF1). CRL4/VPRBP is essential for regulating mammalian oocyte 

survival and reprogramming via activation of TET methylcytosine dioxygenases. 

23387299, 

23453970 

19158078 

16547521 

17543862 

21840486, 

20389280 

23455478 

22578813 

16524876, 

19684112, 

21577200, 

24508459, 

24656772 

16949367, 

18794347, 

20932471, 

23109433 

20223979, 

24292623 

12615916, 

16931761, 

21572435, 

20062082, 

21572435 

15882621, 

16678110 

18381890 

19557001 

21228219 

20551172 

24357321 


CUL5/SOCS1 Dab1 CUL5/SOCS1 is part of an SCF-like ECS (Elongin BC-CUL2/5-SOCS-box 17974915 

protein) E3 ubiquitin ligase complex. CUL5/SOCS1 targets components of the 

Jak/Stat pathway as well as Dab1, a regulator of cortical development, for 


CUL5/SOCS4 EGFR CUL5/SOCS4 is part of an SCF-like ECS (Elongin BC-CUL2/5-SOCS-box protein) 

17997974 

E3 ubiquitin ligase complex. SOCS4 may target components of cytokine 

signal transduction pathways, such as EGF receptor (EGFR) for ubiquitination 

and degradation by the proteasome. 

CUL5/Vif APOBEC3G CUL5/Vif is part of an SCF-like ECS (Elongin BC-CUL2/5-SOCS-box protein) E3 15574592 

ubiquitin ligase complex. Vif targets APOBEC3G and APOBEC3F for ubiquitination 

and degradation by the proteasome. The interaction of Vif with APOBEC3G 

also blocks its cytidine deaminase activity in a proteasome-independent 

manner, 

CUL7/ cyclin D1 CUL7/FBXW8 is an SCF-like E3 ubiquitin ligase complex composed of SKP1, 17205132 

FBXW8 

CUL7, RBX1, GLMN isoform 1, and the substrate recognition component, 

FBXW8. 

DZIP3 H2AK119 DZIP3 is an E3 ubiquitin ligase that blocks transcriptional elongation by 12538761 

ubiquinating H2A at lysine 119. 

E6-AP p53, Dlg E6-AP is also known as UBE3A. E6-AP is a HECT domain E3 ubiquitin ligase 17108031 

that interacts with Hepatitis C virus (HCV) core protein and targets it for 

degradation. The HCV core protein is central to packaging viral DNA and other 

cellular processes. E6-AP also interacts with the E6 protein of the human 

papillomavirus types 16 and 18, and targets the p53 tumor-suppressor protein 

for degradation. 

FANCL FANC D2 FANCL is an ubiquitin ligase protein integral to the DNA repair pathway. 12973351 

HACE1 

HACE1 is an E3 ubiquitin ligase and tumor suppressor. Research has shown 17694067 

that aberrant methylation of HACE1 is frequently found in Wilms’ tumors and 

colorectal cancer. 

HECTD1 

HECTD1 is an ubiquitin E3 ligase required for neural tube closure and normal 17442300 

development of the mesenchyme. 

HECTD2 

HECTD2 is a probable E3 ubiquitin ligase and may act as a susceptibility gene 19214206 

for neurodegeneration and prion disease. 

HECTD3 

HECTD3 is a probable E3 ubiquitin ligase and may play a role in cytoskeletal 

regulation, actin remodeling, and vesicle trafficking. 

18194665 

HECW1 

DVL1, mutant 

SOD1, p53 

HECW1 is also known as NEDL1. HECW1 interacts with p53 and the Wnt 

signaling protein DVL1, and may play a role in p53-mediated cell death in 

neurons. 

HECW2 p73 HECW2 is also known as NEDL2. HECW2 ubiquitinates p73, which is a p53 

family member. Ubiquitination of p73 increases protein stability. 

HERC2 RNF8 HERC2 belongs to a family of E3 ubiquitin ligases involved in membrane 

trafficking events. HERC2 plays a role in the DNA damage response through 

interaction with RNF8. 

HERC3 

HERC3 belongs to a family of E3 ubiquitin ligases involved in membrane trafficking 

events. HERC3 interacts with hPLIC-1 and hPLIC-2 and localizes to the 

late endosomes and lysosomes. 

HERC4 

HERC4 belongs to a family of E3 ubiquitin ligases involved in membrane 

trafficking events. HERC4 is highly expressed in testis and may play a role in 

spermatogenesis. 

HERC5 

HERC5 belongs to a family of E3 ubiquitin ligases involved in membrane 

trafficking events. HERC5 is induced by interferon and other pro-inflammatory 

cytokines and plays a role in interferon-induced ISG15 conjugation during the 

innate immune response. 

HLTF PCNA HLTF is both a helicase and an E3 ubiquitin ligase. HLTF participates in 

postreplication repair (PRR) of damaged DNA by polyubiquitination of 

chromatin-bound PCNA. 

HOIP PKC HOIP is the E3 ubiquitin ligase of the LUBEC (linear ubiquitin chain assembly 

complex) which ubiquitinates signaling proteins, targeting them for proteasomal 

degradation. 

HUWE1 

N-Myc, C-Myc, 

p53, Mcl-1, 

TopBP1 

HUWE1 is also known as Mule. HUWE1 is a HECT domain E3 ubiquitin ligase 

that regulates degradation of Mcl-1 and therefore regulates DNA damage-induced 

apoptosis. HUWE1 also controls neuronal differentiation by destabilizing 

N-Myc, and regulates p53-dependent and independent tumor suppression 

via ARF. 

PhosphoSitePlus® 

A comprehensive online protein modification resource. www.cellsignal.com/exploration 

14684739, 

18223681 

12890487 

20023648 

18535780 

17967448 

16407192, 

16815975 

18316726 

17069764 

15989957 



127




HYD CHK2 HYD is also known as EDD or UBR5. HYD is a regulator of the DNA damage 18073532 

response and is overexpressed in many forms of cancer. 

IBRDC2 p21, Bax IBRDC2 is an E3 ubiquitin ligase involved in the regulation of apoptosis. 12853982 

IBRDC2 expression can be induced by p53 and may target apoptosis related 

proteins p21 and Bax. 

IBRDC3 UCKL-1 IBRDC3 is an E3 ubiquitin ligase involved in the cytolytic activities of hematopoietic 

16709802 

natural killer cells and T cells. 

ITCH MKK4, RIP2, 

Foxp3 

ITCH plays a role in T cell receptor activation and signaling through ubiquitination 

of multiple proteins including MKK4, RIP2, and Foxp3. Loss of ITCH function 

leads to an aberrant immune response and T helper cell differentiation. 

LNX1 NUMB LNX1 is an E3 ubiquitin ligase that plays a role in cell fate determination during 

embryogenesis through regulation of NUMB, the negative regulator of Notch 

signaling. 

LRSAM1 Tsg101 LRSAM is an E3 ubiquitin ligase that mediates intracellular vesicular trafficking 

by monoubiquitination of TSG101. 

Mahogunin 

Mahogunin is an E3 ubiquitin ligase involved in melanocortin signaling. Loss of 

mahogunin function leads to neurodegeneration and loss of pigmentation, and 

may be the mechanism of action in prion disease. 

19737936, 

19592251, 

20108139 

11782429 

15256501 

19737927, 

19524515 

MALIN laforin Malin, also known as NHLRC1, is an E3 ubiquitin ligase that promotes the 15930137 

ubiquitination and proteasomal degradation of misfolded proteins. 

MARCH-I HLA-DRβ MARCH1 is an E3 ubiquitin ligase found on antigen presenting cells (APCs). 19880452 

MARCH1 ubiquitinates MHC class II proteins and downregulates their cell 

surface expression. 

MARCH-II 

MARCH-II is a member of the MARCH family of E3 ubiquitin ligases. It associates 

15689499 

with syntaxin6 in the endosomes and helps to regulate vesicle trafficking. 

MARCH-III 

MARCH-III is a member of the MARCH family of E3 ubiquitin ligases. MARCH- 16428329 

III associates with syntaxin6 in the endosomes and helps to regulate vesicle 

trafficking. 

MARCH-IV MHC class I MARCH-IV is a member of the MARCH family of E3 ubiquitin ligases. MARCH- 14722266 

IV ubiquitinates MHC class I proteins and downregulates their cell surface 

expression. 

MARCH-V DRP1 MARCH-V is a member of the MARCH family of E3 ubiquitin ligases. March-V 16936636 

is located in the mitochondria and aids in the control of mitochondrial morphology. 

MARCH-VI 

MARCH-VI is also known as TEB4 and is a member of the MARCH family of E3 16373356 

ubiquitin ligases. It localizes to the endoplasmic reticulum and participates in 

ER-associated protein degradation. 

MARCH-VII gp190 MARCH-VII is also known as axotrophin. MARCH-VII was originally identified as 19901269 

a neural stem cell gene, but has since been shown to play a role in LIF signaling 

in T lymphocytes through degradation of the LIF receptor subunit, gp190. 

MARCH-VIII 

B7-2, MHC 

class II 

MARCH-VIII is also known as c-MIR. MARCH-VIII causes the ubiquitination/ 

degradation of B7-2, which is a co-stimulatory molecule for antigen presentation. 

MARCH-VIII has also been shown to ubiquitinate MHC class II proteins. 

MARCH-IX ICAM-1, MHC-I MARCH-IX is a member of the MARCH family of E3 ubiquitin ligases. MARCH- 

IX mediates ubiquitination of transmembrane proteins, marking them for 

endocytosis and sorting to lysosomes via multivesicular bodies. 

MARCH-X 

MARCH-X is also known as RNF190. MARCH-X is a member of the MARCH 

family of E3 ubiquitin ligases. MARCH-X may be involved in spermiogenesis. 

MARCH-XI CD4 MARCH-XI is a member of the MARCH family of E3 ubiquitin ligases. MARCH- 

IX mediates ubiquitination of CD4, marking it for endocytosis and sorting to 

lysosomes via multivesicular bodies. 

MDM2 p53 MDM2, an E3 ubiquitin ligase for p53, plays a central role in regulation of 

the stability of p53. Akt-mediated phosphorylation of MDM2 at Ser166 and 

Ser186 increases its interaction with p300, allowing MDM2-mediated ubiquitination 

and degradation of p53. 

MEKK1 c-Jun, Erk MEKK1 is a well known protein kinase of the STE11 family. MEKK1 phosphorylates 

and activates MKK4/7, which in turn activates JNK1/2/3. MEKK1 

contains a RING finger domain and exhibits E3 ubiquitin ligase activity toward 

c-Jun and Erk. 

MGRN1 Tsg101 MGRN1 is an E3 ubiquitin ligase that mediates intracellular vesicular trafficking 

by monoubiquitination of TSG101. 

MIB1 Delta, Jagged Mindbomb homolog 1 (MIB1) is an E3 ligase that facilitates the ubiquitination 

and subsequent endocytosis of the Notch ligands, Delta and Jagged. 

MIB2 Delta, Jagged Mindbomb 2 (MIB2) is an E3 ligase that positively regulates Notch Signaling. 

MIB2 has been shown to play a role in myotube differentiation and muscle 

stability. MIB2 ubiquitinates NMDAR subunits to help regulate synaptic plasticity 

in neurons. 

MID1 PP2A Mid1, also known as Midline-1, is an E3 ubiquitin ligase that may target 

protein phosphatase 2 for ubiquitination and proteasomal degradation. 

16785530 

17174307, 

14722266 

21937444 

17604280 

9153395 

12049732, 

17101801 

17229889 

16000382 

15824097, 

18216171, 

17962190 

11685209 


MKRN1 hTERT, p53, MKRN1 is an E3 ubiquitin ligase that regulates both anti- and pro-apoptotic 15805468 

CDKN1A, FLIP1 functions. 

MycBP2 Fbxo45, TSC2 MycBP2 is an E3 ubiquitin ligase also known as PAM. MycBP2 associates with 

Fbxo45 to play a role in neuronal development. MycBP2 also regulates the 

mTOR pathway through ubiquitination of TSC2. 

NEDD4 

NEDD4 is an E3 ubiquitin ligase highly expressed in the early mouse embryonic 

central nervous system. NEDD4 downregulates both neuronal voltagegated 

Na+ channels (NaVs) and epithelial Na+ channels (ENaCs) in response 

to increased intracellular Na+ concentrations. 

NEDD4L Smad2, PTEN NEDD4L is an E3 ubiquitin ligase highly expressed in the early mouse embryonic 

central nervous system. NEDD4L has been shown to negatively regulate 

TGF-β signaling by targeting Smad2 for degradation. 

NEURL Jagged 1, Delta NEURL is an E3 ubiquitin ligase involved in Notch signaling and neurological 

determination of cell fate. 

OSTM1 Gai3 OSTM1 is an E3 ubiquitin ligase localized to the cell membrane that 

regulates membrane associated G-proteins by ubiquitination and 

proteasomal degradation. 

PARC 

Parkin 

Pael-R, 

CDC-rel, 

PLC-g1,MFN1 

PARC is a cullin family member that acts as a p53-binding cytoplasmic 

anchor protein and is part of an atypical cullin-RING- based E3 ubiquitin ligase 

complex. 

Parkin is an E3 ubiquitin ligase that has been shown to be a key regulator of 

the autophagy pathway. Mutations in Parkin can lead to Parkinson’s Disease. 

PCGF1 H2A, K119 PCGF1 is a component of the polycomb group multiprotein PRC1- like (PcG 

PRC1) complex. PCGF1 is required for PcG PRC1 mediated monoubiquitination 

of H2A Lys119, which is central to the histone code and gene regulation. 

PELI1 TRIP, IRAK PELI1 is an E3 ubiquitin ligase that plays a role in Toll-like Receptor (TLR3 and 

TLR4) signaling to NF-κB via the TRIP adaptor protein. PELI1 has also been 

shown to ubiquitinate IRAK. 

PEX10 Pex5 PEX10 is localized to peroxisome membranes and has been associated with 

several peroxisomal biogenesis disorders. 

PJA1 ELF PJA1 is also known as PRAJA. PJA1 plays a role in downregulating TGF-β 

signaling in gastric cancer via ubiquitination of the Smad4 adaptor protein ELF. 

PJA2 

PJA2 is an E3 ubiquitin ligase found in neuronal synapses. The exact role and 

substrates of PJA2 are unclear. 

RAD18 PCNA RAD18 is an E3 ubiquitin ligase involved in post-replication repair of UVdamaged 

DNA. 

RBCK1 

SOCS6, PKC, 

TAB2/3 

RBCK1 is an E3 ligase that acts as an iron sensor by promoting the ubiquitination 

of oxidized IREB2 in the presence of high iron and oxygen. RBCK1 is a 

component of the LUBAC (linear ubiquitin chain assembly complex). 

RCHY1 p27 Kip1, p53 RCHY1, also known as Pirh2, is an E3 ubiquitin ligase that contributes to the 

regulation of the cell cycle. RCHY1 is primarily associated with the ubiquitination 

and proteasomal degradation of tetrameric p53. 

RFFL p53 RFFL is also known as CARP2 and is an E3 ubiquitin ligase that inhibits endosome 

recycling. RFFL also degrades p53 through stabilization of MDM2. 

RFWD2 MTA1, p53, 

FoxO1 

RFWD2 is also known as COP1. RFWD2 is an E3 ubiquitin ligase that ubiquitinates 

several proteins involved in the DNA damage response and apoptosis 

including MTA1, p53, and FoxO1. 

Rictor SGK1 Rictor interacts with CULlin1-Rbx1 to form an E3 ubiquitin ligase complex and 

promotes ubiquitination and degradation of SGK1. 

RING1 H2A, K119 RING1, also known as RNF1, is an E3 ubiquitin ligase of the polycomb group 

multiprotein PRC1-like (PcG PRC1) complex. RING1 is required for PcG PRC1 

mediated monoubiquitination of H2A Lys119, which is central to the histone 

code and gene regulation. 

RNF2 H2A, K119, 

Geminin 

RNF2, also known as Ring2, is an E3 ubiquitin ligase of the polycomb group 

multiprotein PRC1-like (PcG PRC1) complex. RNF2 is required for PcG PRC 

mediated monoubiquitination of H2A Lys119, which is central to the histone 

code and gene regulation. 

RNF5 JAMP, paxillin RNF5 is also known as RMA5. RNF5 plays a role in ER-associated degradation 

of misfolded proteins and ER stress response through ubiquitination of JAMP. 

RNF5 also plays a role in cell motility and has been shown to ubiquitinate 

paxillin. 

RNF6 

LIM1, Androgen 

receptor 

RNF6 is an E3 ubiquitin ligase involved in the regulation of cell motility and 

differentiation. RNF6 targets LIMK for ubiquitination and degradation, inhibiting 

cytoskeleton stability. 

RNF8 H2A, H2AX RNF8 is a RING domain E3 ubiquitin ligase that plays a role in the repair 

of damaged chromosomes. RNF8 ubiquitinates Histone H2A and H2A.X at 

double-strand breaks (DSBs) which recruits 53BP1 and BRCA1 repair proteins. 

19398581, 

18308511 

9618557, 

9792722 

19917253, 

17218260 

17003037, 

11696324 

12826607 

12526791 

20074049, 

18671761, 

17553932, 

16672220 

18460542 

19734906, 

17675297 

15283676 

16096365 

12036302 

17720710, 

18245774 

17449468, 

16643902, 

12654245, 

18006823, 

18344599 

15229288, 

18382127 

19805145, 

16931761, 

18815134 

20832730 

16359901 

17157253, 

15509584 

19269966, 

12861019 

16204183 

18001824 






RNF11 Smurf2 RNF11 is a required component of a ubiquitin-editing protein complex involved 14562029 

in modifying cellular inflammatory response to LPS and TNF signaling. 

RNF12 CLIM, Ldb1, 

Ldb2 

RNF12, also known as RLIM, is an E3 ubiquitin ligase. RNF12 is involved in 

telomere regulation and X chromosome inactivation. 

12874135, 

11882901 

RNF19 SOD1 RNF19 is also known as Dorfin. Accumulation and aggregation of mutant 19610091 

SOD1 leads to ALS disease. RNF19 ubiquitinates mutant SOD1 protein, causing 

a decrease in neurotoxicity. 

RNF20 Histone H2B RNF20 is also known as BRE1. RNF20 is an E3 ubiquitin ligase that monoubiquitinates 

18832071 

Histone H2B. H2B ubiquitination is associated with areas of 

active transcription. 

RNF34 Caspase-8, -10 RNF34 is also known as RFI. RNF34 inhibits death receptor mediated apoptosis 16596200 

through ubiquitination/degradation of caspase-8 and -10. 

RNF40 Histone H2B RNF40 is also known as BRE1-B. RNF40 forms a protein complex with RNF20 

resulting in the ubiquitination of Histone H2B. H2B ubiquitination is associated 

with areas of active transcription. 

16307923 

RNF41 

RNF111 

ErbB3, BIRC6, 

Parkin 

Smad, SnoN, 

c-Ski 

RNF41 is an E3 ubiquitin ligase that has been implicated in the regulation of 

hematopoietic progenitor cell differentiation. 

RNF111 is an E3 ubiquitin ligase that participates in mesoderm patterning 

by promoting the ubiquitination and proteasomal degradation of downstream 

Smads. 

RNF123 CDKN1B RNF123 is an E3 ubiquitin ligase that functions as part of the KPC complex. 

RNF123 aids in cell cycle regulation by targeting CDKN1B for ubiquitination 

and proteasomal degradation during G1. 

RNF125 

RNF125 is also known as TRAC-1. RNF125 has been shown to positively 

regulate T cell activation. 

RNF128 

RNF128 is also known as GRAIL. RNF128 promotes T cell anergy and may 

play a role in actin cytoskeletal organization in T cell/APC interactions. 

RNF135 RIG-1 RNF135 is an E3 ubiquitin ligase involved in viral innate immunity. RNF135 

targets the cytoplasmic viral nucleic acid receptor RIG-1 for ubiquitination and 

degradation by the proteasome. 

RNF138 TCF/LEF RNF138 is also known as NARF. RNF138 is associated with Nemo-like Kinase 

(NLK) and suppresses Wnt/β-Catenin signaling through ubiquitination/degradation 

of TCF/LEF. 

RNF167 

TSSC5 

(SLC22A18) 

RNF167 may act as an E3 ubiquitin ligase involved in the regulation of kidney 

transporter function. 

RNF168 H2A, H2A.X RNF168 is an E3 ubiquitin ligase that helps protect genome integrity by 

working together with RNF8 to ubiquitinate Histone H2A and H2A.X at DNA 

double-strand breaks (DSB). 

RNF180 Zic2 RNF180 is an E3 ubiquitin ligase involved in neurological development. 

RNF180 targets the ZIC2 transcription factor for polyubiquitination and degradation 

by the proteasome. 

RNF182 ATP6VOC RNF 182 is an E3 ubiquitin ligase that targets ATP6V0C, a component of 

vacuolar ATPase, for polyubiquitination and degradation by the proteasome. 

RNF190 

see MARCH-X 

SCF/β-TrCP 

IκBα, Wee1, 

Cdc25A, 

β-Catenin 

SCF/β-TrCP is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, β-TrCP (also known as 

BTRC). SCF/β-TrCP mediates the ubiquitination of proteins involved in cell cycle 

progression, signal transduction, and transcription. SCF/β-TrCP also regulates 

the stability of β-catenin and participates in Wnt signaling. 

SCF/FBXW2 GCMa SCF/FBXW2 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 

CUL1-F-box protein) and the substrate recognition component, FBXW2. SCF/ 

FBXW2 promotes ubiquitination of GCMa which is important for trophoblast 

cell differentiation. 

SCF/FBXW5 SASS6, Eps8 SCF/FBXW5 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXW5. SCF/FBXW5 

mediates the ubiquitination of SASS6, preventing centriole duplication. 

SCF/FBXW7 Cyclin-E, c- 

Myc, c-Jun 

SCF/FBXW8 

IRS-1, TBC1D3, 

Cyclin D1, 

IGFBP2, HPK1 

SCF/FBXW7 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 


mediates the ubiquitination of proteins involved in cell cycle progression, signal 

transduction, and transcription. Target proteins for SCF/FBXW7 include the 

phosphorylated forms of c-Myc, Cyclin E, Notch intracellular domain (NICD), 

and c-Jun. Research has found that defects in FBXW7 may be a cause of many 

types of human cancers including T-ALL, colon cancer and breast cancer. 

SCF/FBXW8 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 


plays critical roles in various cellular processes such as cell cycle progression, 

cell differentiation, development, and growth factor signaling pathway. 

14765125, 

12411582, 

18541373 

18451154, 

14657019, 

17591695 

15531880 

17990982 

19833735 

19017631 

16714285 

16314844 

19203579 

18363970 

18298843 

10230406, 

15070733, 

14603323, 

10339577 

15640526 

18381890, 

23314863 

11533444, 

15150404, 

16023596 

23142081, 

23029530, 

17205132, 

24362026 


SCF/ 

FBXW10 

CBX1, CBX5 SCF/FBXW10 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 


FBXW10 contributes to gene expression by degradation of heterochromatin 

components CBX5 and CBX1. 

20498703 

SCF/ 

FBXW15 

SCF/Skp2/ 

FBXL1 

SCF/FBXL2 

HBO1 

p27, p21, 

FoxO1 

Aurora B, p85b, 

APP, Cyclin D2 

SCF/FBXW15 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 23319590 


FBXW15 controls DNA replication via degradation of the origin recognition 

complex protein HBO1. 

SCF/Skp2 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1-Fbox 

protein) and the substrate recognition component, Skp2. SCF/Skp2 medi- 

10559916 

15668399, 

ates the ubiquitination of proteins involved in cell cycle progression (specifically 

the G1/S transition), signal transduction and transcription. Target proteins for 

SCF/Skp2 include the phosphorylated forms of p27Kip1, p21Waf1/Cip1, and 

FoxO1. 

SCF/FBXL2 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXL2. SCF/FBXL2 is 

involved in cell cycle regulation, growth factor signaling and synapse formation. 

SCF/FBXL3 CRY1, CRY2 SCF/FBXL3 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXL3. SCF/FBXL3 

mediates circadian clock function by ubiquitination and subsequent degradation 

of CRY1 and CRY2. 

SCF/FBXL4 

SCF/FBXL5 

KDM4A/JM- 

JD2A 

IRP2, 

p150(Glued) 



regulates chromatin structure through degradation of the lysine demethylase 

KDM4A/JMJD2A. 

SCF/FBXL5 is an ubiquitin ligase complex also known as SCF (SKP1-cullin-Fbox). 

FBXL5 is an F-box protein that functions as an iron sensor by promoting 

the ubiquitination and subsequent degradation of IREB2/IRP2 under high iron 

and oxygen conditions. 

SCF/FBXL7 Aurora A SCF/FBXL7 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 


colocalizes with and promotes degradation of Aurora A during mitosis leading 

to impaired cell proliferation. 

SCF/FBXL10 

p15, c-Fos, 

Ink4a, RipK3 



plays critical roles in cell proliferation, senescence and stem cell self-renewal. 

SCF/FBXL11 p65 FBXL11 is a lysine demethylase that regulates NF-κB signaling pathway through 

p65 demethylation. A substrate for ubiquitination has yet to be identified. 

SCF/FBXL12 

SCF/FBXL14 

SCF/FBXL15 

SCF/FBXL19 

Ku80, 

Calmodulin 

kinase I 

SNAIL1, SLUG, 

Mkp3 

Timeless, 

SMURF1, 

SMURF2 

ST2L, Rac1, 

RhoA 



is involved in regulating cell cycle progression and DNA damage response. 


F-box protein) and the substrate recognition component,FBXL14. SCF/FBXL14 

is associated with metastasis and cancer development. 


F-box protein) and the substrate recognition component, FBXL15. FBXL15 targets 

negative regulators of the BMP signaling pathway, including SMURF1 and 

SMURF2, for ubiquitination and subsequent proteasomal degradation. FBXL15 

is required for dorsal/ventral pattern formation and bone mass maintenance. 

SCF/FBXL15 also targets the Drosophila circadian clock protein timeless. 



is an important regulator of cell proliferation and migration, cytoskeleton 

reorganization, and pulmonary inflammation. 

SCF/FBXL20 RIM1 SCF/FBXL20 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXL20. FBXL20 is 

localized to the synapse and its regulation of RIM1 by ubiquitination may play a 

role in neural transmission. 

SCF/FBXL21 CRY SCF/FBXL21 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 

CUL1-F-box protein) and the substrate recognition component, FBXL21. SCF/ 

FBXL21 maintains circadian clock rhythms through controlling CRY turnover 

cooperatively with SCF/FBXL3. 


A comprehensive online protein modification resource. www.cellsignal.com/exploration 

23928698, 

23604317, 

22399757, 

22323446 

17463251, 

17463252, 

17462724 

21757720 

19762597, 

19762596 

22306998 

18836456, 

21252908, 

21540074, 

22825849 

20080798 

23324393, 

23707388 

19955572, 

16887825, 

22410791 

16794082, 

21572392 

22660580, 

23512198, 

23871831 

17803915 

23452856, 

23452855, 

18953409 



131




SCF/FBXL22 ACTN/FLNC SCF/FBXL22 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXL22. FBXL22 

is exclusively expressed in cardiomyocytes, and promotes ubiquitination and 

degradation of sarcomeric proteins, α-actinin-2 (ACTN) and Filamin C (FLNC). 

22972877 

SCF/FBXO1 CP110, RRM2 SCF/FBXO1 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXO1. FBXO1 

targets CP110 for ubiquitination and subsequent proteasomal degradation during 

cell cycle G2 phase, thereby inhibiting centrosome reduplication. 

SCF/FBXO2 Pre-integrin 

β-1, CFTR, 

Connexin 26, 

NR1, SHPS-1, 

UL9 

SCF/FBXO3 HIPK2, p300, 

Fbxl2 

SCF/FBXO4 

SCF/FBXO6 

SCF/FBXO7 

TERF1, Cyclin 

D1 

Chk1, ATRN, 

TCRa, TFRC 

BIRC2, DL- 

GAP5, CD43 

SCF/FBXO2 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXO2 (NFB42). 

FBXO2 targets misfolded glycoproteins for ubiquitination and proteasomal 

degradation by recognition of sugar chains in the endoplasmic reticulumassociated 

degradation (ERAD) pathway. 



targets HIPK2 and p300 for ubiquitination and rapid degradation by the 

proteasome. The inclusion of PML in a complex with SCF/FBXO3, HIPK2, and 

p300 delays degradation of HIPK2 and allows synergistic activation of p53/ 

TP53-dependent transactivation. 


F-box protein) and the substrate recognition component, FBXO4. FBXO4 may 

play a role in telomere homeostasis by recognition of TERF1 and promotion of 

its ubiquitination together with UBE2D1. 



targets misfolded glycoproteins for ubiquitination and proteasomal degradation 

by recognition of sugar chains in the endoplasmic reticulum-associated 

degradation (ERAD) pathway. FBXO6 also targets the kinase Chk1, a cell cycle 

regulator involved in entry into mitosis. 


F-box protein) and the substrate recognition component, FBXO7. SCF/FBXO7 

targets BIRC2 (cIAP1), an inhibitor of apoptosis, and DLGAP5, a cell cycle 

regulator, for ubiquitination and proteasomal degradation. 

SCF/FBXO8 Arf6 SCF/FBXO8 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 


mediates the ubiquitination of Arf6. SCF/FBXO8 may function as a guanine 

nucleotide exchange factor (GEF) that activates ARF G proteins. Loss of FBXO8 

correlates with poor survival in hepatocellular carcinoma. 

SCF/FBXO9 Tel2, Tti1 SCF/FBXO9 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 

CUL1-F-box protein) and the substrate recognition component, FBXO9. Three 

isoforms of the human protein are produced by alternative splicing, and it is 

linked to promoting survival in multiple myeloma. 

SCF/FBXO10 Bcl2 SCF/FBXO10 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 

CUL1-F-box protein) and the substrate recognition component, FBXO10. SCF/ 

FBXO10 induces apoptosis in B-cells through degrading Bcl2. 

SCF/FBXO11 Cdt2, Bcl6, p53 SCF/FBXO11 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 

CUL1-F-box protein) and the substrate recognition component, FBXO11. 

SCF/FBXO11 promotes the neddylation of p53 to suppress its transcriptional 

activity, and crosstalks with the cullin 4-RING ubiquitin ligase, CRL4-Cdt2, 

to control cell cycle progression. Mutations and deletions in FBXO11 lead to 

BCL6 stabilization and B-cell lymphoma. FBXO11 is also regarded as a haploinsufficient 

tumor suppressor gene. 

SCF/FBXO15 

P-glycoprotein/ 

ABCB1 

SCF/FBXO15 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 


FBXO15 activity may affect anticancer drug resistance through controlling the 

expression of P-glycoprotein (P-gp)/ABCB1 on cancer cell surfaces. 

20596027, 

22632967 

12140560, 

15809437, 

14701835, 

17494702 

18809579, 

23542741, 

18809579 

17081987, 

19645770, 

17081987 

19716789, 

22268729, 

12939278 

16510124, 

21652635 

18094045 

23263282 

23431138 

23478445, 

23478441, 

23892434, 

22113614, 

17098746 

23465077 

SCF/FBXO17 Arf1 SCF/FBXO17 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 19836238 

CUL1-F-box protein) and the substrate recognition component, FBXO17. In 

fission yeast, SCF/FBXO17 binds the Arf1 transcription factor and promotes 

Arf degradation to suppress stress-related gene expression. Stress-induced Arf 

phosphorylation dissociates Arf interaction with FBXO17. 

SCF/FBXO22 KDM4A SCF/FBXO22 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 


FBXO22 is involved in transcriptional control through degradation of lysine 

demethylase KDM4A. 

21768309 

SCF/FBXO25 

NKX2.55, Isl1, 

Hand1, Elk-1 



is a cardiac-specific F-box protein. SCF/FBXO25 maintains cardiac protein 

homeostasis through degrading NKX2.5, Isl1, and Hand1, and suppresses 

mitogenic response by downregulating Elk-1. 

21596019, 

23940030 


SCF/FBXO31 Cyclin D1, 

Par6c 


F-box protein) and the substrate recognition component, FBXO31 (FBXO14). 

SCF/FBXO31 targets phosphorylated cyclin D1 for ubiquitination and degradation 

by the proteasome, resulting in G1 cell cycle arrest. 

19412162 

SCF/FBXO32 

DUSP1, eIF3-f, 

MyoD, BK-β(1), 

FOXO1 


F-box protein) and the substrate recognition component, FBXO32 (MAFbx). 

FBXO32 is a cardiac-specific F-box protein. SCF/FBXO32 maintains cardiac 

protein homeostasis. FBXO32 promoter is highly methylated in pediatric softtissue 

sarcoma. 

SCF/FBXO33 YB-1 SCF/FBXO33 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 

CUL1-F-box protein) and the substrate recognition component, FBXO33. 

SCF/FBXO33 is involved in gene expression and protein translation through 

degradation of Y-box protein YB-1. 

SCF/FBXO40 IRS-1 SCF/FBXO40 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXO40. FBXO40 is 

a skeletal muscle-specific F-box protein. SCF/FBXO40 controls muscle size by 

regulating IRS-1 protein stability. 

SCF/FBXO42 p53 SCF/FBXO42 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 


targets p53/TP53 for ubiquitination and degradation by the proteasome. 

SCF/FBXO44 BRCA1 SCF/FBXO44 is an E3 ubiquitin ligase complex composed of SCF (SKP1- 


FBXO44 activity may contribute to breast cancer development through targeting 

BRCA1 for proteasomal degradation. 

SCF/FBXO45 UNC13A, p73 SCF/FBXO45 is an E3 ubiquitin ligase complex composed of SCF (SKP1-CUL1- 

F-box protein) and the substrate recognition component, FBXO45. SCF/FBXO45 

aids in the regulation of neurotransmission at mature neurons by targeting 

UNC13A for ubiquitin dependent degradation by the proteasome. FBXO45 also 

targets the apoptotic protein p73 for ubiquitination and degradation. 

SHPRH PCNA SHPRH is an E3 ubiquitin ligase that plays a role in DNA replication through 

ubiquitination of PCNA. PCNA ubiquitination prevents genomic instability from 

stalled replication forks after DNA damage. 

SIAH1 

β-catenin, Bim, 

TRB3 

SIAH1 is an E3 ubiquitin ligase that plays a role in inhibition of Wnt signaling 

through ubiquitination of β-catenin. SIAH1 has also been shown to promote 

apoptosis through upregulation of Bim, and to ubiquitinate the signaling adaptor 

protein TRB3. 

SIAH2 HIPK2, PHD1/3 SIAH2 is an E3 ubiquitin ligase that plays a role in hypoxia through ubiquitination 

and degradation of HIPK2. SIAH2 also ubiquitinates PHD1/3, which 

regulates levels of HIF-1α in response to hypoxia. 

SMURF1 

Smad1/5, 

RhoA, MEKK2 

SMURF1 is an E3 ubiquitin ligase that interacts with BMP pathway Smad 

effectors, leading to Smad protein ubiquitination and degradation. Smurf1 

negatively regulates osteoblast differentiation and bone formation in vivo. 

SMURF2 Smads, Mad2 SMURF2 is an E3 ubiquitin ligase that interacts with Smads from both the BMP 

and TGF-β pathways. SMURF2 also regulates the mitotic spindle checkpoint 

through ubiquitination of Mad2. 

SYVN1 

ERAD, Pael- 

R,p53, IRE-1 

SYVN1 is an E3 ubiquitin ligase involved in the ER-associated degradation 

(ERAD) pathway. SYVN1 targets misfolded proteins and appropriately folded 

short-lived proteins for ubiquitination and degradation by the proteasome. 

TOPORS p53, NKX3.1 TOPORS is an E3 ubiquitin ligase and a SUMO ligase. TOPORS ubiquitinates 

and sumoylates p53, which regulates p53 stability. TOPORS has also been 

shown to ubiquitinate the tumor suppressor NKX3.1. 

TRAF2 

Rip1, other 

TRAFs 

TRAF2 is a weak E3 ubiquitin ligase that acts as a component of several 

ubiquitination complexes. TRAF2 ligase activity is activated in the presence 

of cytoplasmic sphingosine-1-phosphate. TRAF2 is a major regulator of the 

apoptosis and cell survival machinery. 

TRAF6 NEMO, Akt1 TRAF6 is an E3 ubiquitin ligase that functions as an adaptor protein in IL-1R, 

CD40, and TLR signaling. TRAF6 promotes NF-κB signaling through K63 

polyubiquitination of IKK, resulting in IKK activation. TRAF6 has also been 

shown to ubiquitinate Akt1, causing its translocation to the cell membrane. 

TRAF7 

TRAF7 is an E3 ubiquitin ligase and SUMO ligase that functions as an adaptor 

protein in TNF Receptor and TLR signaling. TRAF7 has been shown to be capable 

of self-ubiquitination and plays a role in apoptosis via MEKK3-mediated 

activation of NF-κB. 

TRIAD3 TLRs, RIP1 Triad 3 is an E3 ubiquitin ligase found in peripheral blood leukocytes of the immune 

system that regulates antiviral and cytokine induced cellular responses. 

TRIM8 SOCS-1 TRIM8 is an E3 ubiquitin ligase that regulates cytokine induced signal 

transduction by targeting SOCS1 for ubiquitination and degradation by the 

proteasome. 

22586590, 

21454680, 

19073596, 

24213577 

16797541 

22033112 

19509332 

23086937 

19581926 

18719106 

16413921, 

19775288, 

18276110 

19043406, 

15210114 

10458166, 

15820682 

11158580, 

18852296 

14593114, 

17059562, 

17170702, 

18369366 

19473992, 

18077445 

11909853, 

15175328 

19713527, 

11057907 

15001576 

15107846, 

16968706 

12163497 






TRIM11 Humanin, 

ARC105, Pax6 

TRIM11 is an E3 ubiquitin ligase that may promote the degradation of insoluble 

ubiquitinated proteins. TRIMM11 may also aid in anti-viral cellular functions. 

12670303, 

16904669, 

18628401 

TRIM13 

TRIM13 is an E3 ubiquitin ligase that targets membrane and secretory proteins 17314412 

for ubiquitination and proteasomal degradation in the endoplasmic reticulumassociated 

degradation (ERAD) pathway. 

TRIM21 IgG1 HC, IRF3 TRIM21 is an E3 ubiquitin ligase involved in intracellular antibody-mediated 

degradation of viral components by the proteasome. 

TRIM25 RIG-1 TRIM25 is an E3 ubiquitin ligase involved in viral innate immunity. TRIM25 

targets the cytoplasmic viral nucleic acid receptor RIG-1 for ubiquitination and 

degradation by the proteasome. 

TRIM32 actin, piasy TRIM32 is an E3 ubiquitin ligase involved in viral lysosome related vesicle 

trafficking. TRIM32 targets DTNBP1 for ubiquitination and degradation by the 

proteasome. TRIM32 may also mediate the activity of HIV Tat proteins. 

TRIM33 Smad4 TRIM33 is an E3 ubiquitin ligase involved in the regulation of the TGF-b/ BMP 

signaling pathway. TRIM33 targets SMAD4 for ubiquitination, nuclear exclusion, 

and proteasomal degradation. 

TRIM41 PKC TRIM41 is an E3 ubiquitin ligase that targets protein kinase C for ubiquitination 

and proteasomal degradation. 

18022694, 

18641315 

12075357 

14578165, 

16243356 

15820681 

17893151 

TRIM54 TRIM54 is an E3 ubiquitin ligase that may target and stabilize microtubules. 15967462 

TRIM55 

TRIM55 is an E3 ubiquitin ligase that may regulate gene expression and 15967462 

protein turnover in muscle cells 

TRIM63 Troponin I, TRIM63 is also known as Murf-1. TRIM63 is a muscle-specific E3 ubiquitin ligase 19506036 

MyBP-C, whose expression is upregulated during muscle atrophy. TRIM63 has been shown 

to ubiquitinate several important muscle proteins including troponin I, MyBP-C, and 

MyLC1/2. 

MyLC1/2 

TRIM63 is also known as Murf-1. TRIM63 is a muscle-specific E3 ubiquitin 19506036 

ligase whose expression is upregulated during muscle atrophy. TRIM63 has 

been shown to ubiquitinate several important muscle proteins including 

troponin I, MyBP-C, and MyLC1/2. 

UBE3B 

UBE3B is an E3 ubiquitin ligase identified through sequence analysis. The 12837265 

specific substrates and cellular function of UBE3B is currently unknown. 

UBE3C 

UBE3C is an E3 ubiquitin ligase also known as KIAA10. UBE3C is highly expressed 

in muscle and may interact with the transcriptional regulator TIP120B. 

12692129 

UBR1 

UBR1 is an E3 ubiquitin ligase responsible for proteasomal degradation of 

misfolded cytoplasmic proteins. UBR1 has also been shown to be a ubiquitin 

ligase of the N-end rule proteolytic pathway, which regulates degradation of 

short-lived proteins. 

UBR2 Histone H2A UBR2 is an E3 ubiquitin ligase that has been shown to ubiquitinate histone 

H2A, resulting in transcriptional silencing. UBR2 is also part of the N-end rule 

proteolytic pathway. 

19041308, 

17962019 

20080676, 

19008229 

UHRF1 Histone H3 UHRF1 is an epigenetic regulator that is also a putative E3 ubiquitin ligase. 14993289 

UHRF2 PCNP UHRF2 is also known as NIRF. UHRF2 is a nuclear protein that may regulate 

cell cycle progression through association with Chk2. UHRF2 also ubiquitinates 

PCNP and has been shown to play a role in degradation of nuclear aggregates 

containing polyglutamine repeats. 

VHL HIF-1α VHL is the substrate recognition component of the ECV (Elongin B/C, CULlen-2, 

VHL) E3 ubiquitin ligase complex responsible for degradation of the 

transcription factor HIF-1α. Ubiquitination and degradation of HIF-1α takes 

place only during periods of normoxia, but not during hypoxia, thereby playing 

a central role in the regulation of gene expression by oxygen. 

VPS18 SNK VPS18 is an E3 ubiquitin ligase that regulates intracellular vesicle trafficking. 

VPS18 may also regulate the POLO-like kinase SNK during the cell cycle. 

WWP1 ErbB4 WWP1 is an E3 ubiquitin ligase commonly found to be overexpressed in breast 

cancer. WWP1 has been shown to ubiquitinate and degrade ErbB4. Interestingly, 

the WWP1 homolog in C. elegans was found to increase life expectancy 

in response to dietary restriction. 

WWP2 oct-4, PTEN WWP2 is an E3 ubiquitin ligase that has been shown to ubiquitinate/degrade 

the stem cell pluripotency factor Oct-4. WWP2 also ubiquitinates the transcription 

factor EGR2 to inhibit activation-induced T cell death. 

ZNRF1 

ZNRF1 is an E3 ubiquitin ligase highly expressed in neuronal cells. ZNRF1 is 

found in synaptic vesicle membranes and may regulate neuronal transmissions 

and plasticity. 


A comprehensive online protein modification resource. www.cellsignal.com/cstpsp 

15178429, 

14741369, 

19218238 

11292862 

16203730 

19561640, 

19553937 

19274063, 

19651900, 

21532586 

14561866 

DUB Substrate Function PMID 

ATXN3 

ATXN3L 

BRCC36 

COPS5 

COPS6 

RAD23A, 

RAD23B, 

STUB1/CHIP 

FAM175A/ 

Abraxas 

TP53, MIF, 

JUN, UCHL1, 

ESR1, 

RanBP9 

TP53, MIF, 

c-Jun, 

UCHL1 

ATXN3 is a transcriptional regulation deubiquitination enzyme that preferentially 17696782 

displays ubiquitin isopeptidase activity toward long, four or greater, ubiquitin chains. 

ATXN3-like is a deubiquitination enzyme that displays ubiquitin isopeptidase 21118805 

activity toward K48- and K63-linked chains. 

BRCC36 (BRCC3) is a deubiquitination enzyme that preferentially displays ubiquitin 14636569, 

isopeptidase activity toward K63-linked chains. BRCC36 targets K63-linked ubiquitin 

chains on H2A and H2X at the site of DNA double strand breaks as a component 

16707425 

of the BRCA complex. 

COPS5 (CSN5) is the protease subunit of the COP9 signalosome complex (CSN), a 

key regulator of the ubiquitin conjugation pathway. COPS5 is essential for the CSN 

isopeptidase activity responsible for deneddylation of cullin-RING E3 ubiquitin ligase 

complexes. 

COPS6 is a component of the COP9 signalosome complex (CSN) which is involved 

in several cellular and developmental processes. The CSN complex regulates the 

ubiquitin (Ubl) conjugation pathway through the deneddylation of the cullin subunits 

of SCF-type E3 ligase complexes. This decreases the Ubl ligase activity of SCF-type 

complexes such as SCF, CSA, or DDB2. 

9535219, 

11285227, 

23926111 

11337588, 

12732143, 

12628923 

USP17L2 CDC25A USP17L2 is a deubiquitination enzyme that preferentially displays ubiquitin 14699124, 

isopeptidase activity toward CDC25A, preventing CDC25A degradation and allowing 20228808 

cell cycle progression. 

eIF3F Notch eIF3F deubiquitinates activated Notch1 promoting its nuclear import, thereby acting 21124883 

as a positive regulator of Notch signaling. 

eIF3H 

eIF3H is involved in various stps of the initiation of protein synthesis as a component 16766523 

of the eukaryotic translation initiation factor 3 (eIF-3) complex. 

JOSD1 

JOSD1 is a deubiquitination enzyme that displays low ubiquitin isopeptidase activity 21118805 

in vitro. 

JOSD2 

MPND 

MYSM1 

H2A, 

E4BP4, 

GFI1 

JOSD2 is a deubiquitination enzyme that displays ubiquitin isopeptidase activity 

toward K63-linked chains, and to a lesser extent K48-linked chains. 

MPND is an MPN domain and JAMM motif-containing protein with predicted 

ubiquitin isopeptidase activity. 

MYSM1 is a deubiquitinating enzyme that acts as a transcriptional co-activator by 

directing preferential ubiquitin isopeptidase activity toward monoubiquinated H2A in 

hyperacetylated nucleosomes. 

OTU1 VCP OTU1, also known as YOD1, is a deubiquitination enzyme that displays ubiquitin 

isopeptidase activity toward K48- and K63-linked polyubiquitin or di-ubiquitin 

chains. OTU1 is a part of the endoplasmic reticulum-associated degradation (ERAD) 

pathway for misfolded lumenal proteins. 

OTUB1 

RNF128, 

UbcH5, 

Smad2/3, 

c-IAP 

OTUB1 is a deubiquitination enzyme that preferentially displays ubiquitin isopeptidase 

activity toward polyubiquitinated K48-linked chains. OTUB1 regulates protein 

turnover by preventing degradation and also plays a unique role in the regulation 

of T cell anergy. Furthermore, OTUB1 regulates p53 stability and activity via noncanonical 

inhibition of the MDM2 cognate Ub-conjugating enzyme (E2) UbcH5. 

OTUB1 also inhibits the ubiquitination of phospho-SMAD2/3 by binding to and 

inhibiting the E2 ubiquitin-conjugating enzymes independent of its catalytic activity. 

OTUB1 regulates NF-κB and MAPK signaling pathways as well as TNF-dependent 

cell death by modulating c-IAP1 stability. 

OTUB2 RNF-8 OTUB2 is a deubiquitination enzyme that displays ubiquitin isopeptidase activity 

toward K48- and K63-linked chains. OTUB2 regulates protein turnover by preventing 

degradation. OTUB2 fine-tunes the speed of DSB-induced ubiquitination. 

OTUD1 

OTUD1 is a member of the deubiquitinating enzyme ovarian tumor domain (OTU) 

superfamily. 

OTUD3 

OTUD3 is a member of the deubiquitinating enzyme ovarian tumor domain (OTU) 

superfamily. 

OTUD4 XPC OTUD4 is a member of the deubiquitinating enzyme ovarian tumor domain (OTU) 

superfamily. 

OTUD5 

OTUD6A 

OTUD6B 

OTUD7A/ 

Cezanne 2 

OTUD7B/ 

Cezanne 

OTUD7C/ 

A20 

TRAF3, 

p53 

TRAF6, 

TRAF3 

NAF1, 

TAX1BP1, 

TRAF2 

OTUD5 is a deubiquitination enzyme that displays ubiquitin isopeptidase activity 

toward K48- and K63-linked chains. OTUD5 negatively regulates type I interferon 

(IFN) production by deubiquitination of TRAF3. 

OTUD6A is a member of the deubiquitinating enzyme ovarian tumor domain (OTU) 

superfamily. 

OTUD6B is a member of the deubiquitinating enzyme ovarian tumor domain (OTU) 

superfamily. 

OTUD7A is a deubiquitination enzyme that displays ubiquitin isopeptidase activity 

toward K48- and K63-linked chains. 

OTUD7B is a deubiquitination enzyme that displays ubiquitin isopeptidase activity 

toward K48- and K63-linked chains. OTUD7B negatively regulates NF-κB. 

OTUD7C is a ubiquitination-editing enzyme that displays ubiquitin isopeptidase activity 

toward K63-linked chains and ubiquitination of K48-linked chains. OTUD7C is 

an essential regulator of inflammatory signaling pathways in the lymphoid system. 

17696782, 

21118805 

17707232, 

24062447, 

24014243 

19818707 

12704427, 

14661020, 

24403071, 

24071738, 

23524849, 

22124327 

12704427, 

18954305, 

24560272 

17991829 

17991829 

17991829, 

24366067 

17991829, 

24143256 

17991829 

17991829 

12682062 

11463333, 

23334419 

9882303, 

14748687 

Deubiquitinase 

Table 

This table provides a list of 

deubiquitinases (DUBs), along with 

their substrates (when known) and 

corresponding references. 



135




OTULIN Met-1 

polyubiquitin 

OTULIN disassembles Met1-Ub which is important for reducing Met1-Ub accumulation 

after stimulation of nucleotide-oligomerization domain-containing protein 2 

(NOD2). Depletion of OTULIN alters signaling downstream of NOD2. 

23806334 

POH1 

PRPF8 

SNRP116, 

WDR57/ 

SPF38 

POH1 is the metalloprotease deubiquitination enzyme component of the 26S 

proteasome that displays ubiquitin isopeptidase activity toward K63-linked chains. 

PRPF8 is a member of the deubiquitinating enzyme metalloprotease JAMM domain 

superfamily. PRPF8 is known to be a central component of the spliceosome, while 

PRPF8 ubiquitin isopeptidase activity is controversial. 

PSMD7 TRIM5 PSMD7 is a regulatory subunit of the 26S proteasome, and is involved in the ATPdependent 

degradation of ubiquitinated proteins. 

STAMBP 

STAMB- 

PL1 

CXCR4, 

EGFR, ErbB2 

STAM-binding protein (STAMBP or AMSH) is an endosomal deubiquitination enzyme 

that preferentially displays ubiquitin isopeptidase activity toward K63-linked chains. 

STAM-binding protein-like 1 (STAMBPL1 or AMSHLP) is a deubiquitination enzyme 

that preferentially displays ubiquitin isopeptidase activity toward K63-linked chains. 

TRABID TRAF6, APC TRABID is a deubiquitination enzyme that preferentially displays ubiquitin isopeptidase 

activity toward K63-linked chains. TRABID acts as a positive regulator of the 

Wnt signaling pathway by deubiquitinating APC protein, a Wnt signaling pathway 

negative regulator. 

UCHL1 

UCHL2/ 

BAP1 

COPS5, 

CHT, NCAM, 

β-Catenin 

BRCA1, 

HCFC1 

UCHL1 is a member of the ubiquitin C-terminal hydrolase (UCH) deubiquitinase 

superfamily. UCHL1 functions as a ubiquitin hydrolase involved in the processing of 

both ubiquitin precursors and ubiquitinated substrates, generating free monomeric 

Ub. UCHL1 may play a role in regulating cholinergic function through CHT ubiquitination 

and degradation. 

UCHL1/Bap1 is a member of the ubiquitin C-terminal hydrolase (UCH) deubiquitinase 

superfamily. UCHL1/ Bap1 is a BRCA1-associated, nuclear localized ubiquitin 

hydrolase that suppresses cell growth. 


9374539, 

19214193 

2139226, 

8702566 

22078707, 

7755639 

18758443, 

20159979, 

22800866 

18758443 

18281465, 

21834987 

9790970, 

24525247, 

23061666, 

22641175 

9528852 

UCHL3 ENAC UCHL3 is a member of the ubiquitin C-terminal hydrolase (UCH) deubiquitinase 

superfamily. UCHL3 functions as a ubiquitin hydrolase involved in the processing of 

both ubiquitin precursors and ubiquitinated substrates, generating free monomeric 

Ub. UCHL3 shows dual specificity toward both ubiquitin (Ub) and NEDD8, a Ub-like 

molecule. 

2530630 

UCHL5 

USP1 

USP2 

TGF-β 

Receptor I 

FANCD2, 

PCNA, 

PHLPP1 

CCND1, 

PER1 

USP3 H2A, Rig-I , 

H2A, γH2A.X 

USP4 

USP5/ 

ISOT 

ADORA2A, 

RB1, 

Rig-I, RIP1, 

TRAF2, 

TRAF6, 

PDK1 

p53, 

TRIML1 

UCHL5 is a member of the ubiquitin C-terminal hydrolase (UCH) deubiquitinase 

superfamily. UCHL5 is the deubiquitination enzyme component of the 19S regulatory 

subunit of the 26S proteasome that displays ubiquitin isopeptidase activity 

toward K48-linked chains. 

USP1 is a member of the ubiquitin-specific processing protease (USP/USB) deubiquitinase 

superfamily. USP1 is a negative regulator of DNA repair machinery. 


superfamily. USP2 is characterized by its C19 peptidase activity, which is 

involved in ubiquitin recycling and in the disassembly of various forms of polymeric 

ubiquitin and ubiquitin-like protein complexes. USP2 is also a core component of 

circadian rhythm machinery. 

USP3 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP3 deubiquitinates monoubiquitinated histone H2A 

and H2B. USP3 is required for proper progression through S phase and subsequent 

mitotic entry. 


superfamily. USP4 is a proto-oncogene that deubiquitinates target proteins 

such as the receptor ADORA2A and TRIM21 and plays a role in the regulation of 

quality control in the ER. 


deubiquitinase superfamily. USP5 preferentially cleaves branched and K48-linked 

polymers. USP5 binds linear and K63-linked polyubiquitin with a lower affinity. 

Knock-down of USP5 causes the accumulation of p53/TP53 and an increase in 

p53/TP53 transcriptional activity. 

USP6 NF-κB USP6 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP6 exhibits an ATP-dependent C-terminal isopeptidase 

activity. 

USP7/ 

HAUSP 

FOXO4, 

PTEN p53, 

MDM2, 

Tip60, MCM, 

FoxP3 , 

UBE2E1, 

NF-κB, 

Aurora A 

USP7, also known as herpes virus-associated ubiquitin-specific protease (Hausp), 

is a member of the ubiquitin-specific processing protease (USP/USB) deubiquitinase 

superfamily. USP7 deubiquitinates target proteins such as FoxO4, p53/TP53, 

MDM2, PTEN and DAXX. USP7 is Involved in cell proliferation during early embryonic 

development and also plays a role in the regulation of early adipogenesis. 

16906146, 

18922472, 

23500140 

15694335, 

16531995, 

22426999 

17290220, 

19917254, 

19838211, 

23213472 

17980597, 

24366338, 

24196443 

7784062, 

16316627, 

23388719, 

23313255, 

22029577, 

22347420 

19098288 

20418905, 

22081069 

11923872, 

14506283, 

15053880, 

23775119, 

24190967, 

23973222, 

23603909, 

23267096, 

23348568 


USP8/ 

UBPY 

USP9X 

EPS15, 

CLOCK, 

HIF-1α, Smo 

SMAD4, 

MARK4, 

NUAK1, 

BIRC5/ 

survivin, 

Smurf1, 

Mcl-1, ERG, 

Bcl10, PEX5 


deubiquitinase superfamily. USP8 is an essential growth-regulated enzyme that is 

indispensible for cell proliferation and survival. USP8 regulates endosomal ubiquitin 

dynamics, cargo sorting, membrane traffic at early endosomes, and maintenance 

of EGFR stability. In normoxia, USP8 maintains HIF1α and HIF transcriptional output 

which is essential for endosome-mediated ciliogenesis. 

USP9X is an X-linked member of the ubiquitin-specific processing protease 

(USP/USB) deubiquitinase superfamily. USP9X hydrolyzes both ‘Lys-29’- and 

‘Lys-33’-linked polyubiquitin chains. USP9X functions to regulate cell-cell contact 

interactions, TGF-β/BMP signaling, chromosome alignment and segregation, and 

specifically deubiquitinates monoubiquitinated Smad4. Moreover, USP9X is a 

mTORC1 and mTORC2 binding partner that negatively regulates mTOR activity and 

skeletal muscle differentiation. 

USP9Y SMAD4 USP9Y is a Y-linked member of the ubiquitin-specific processing protease (USP/ 

USB) deubiquitinase superfamily required for sperm production. USP9Y functions to 

regulate TGF-β/BMP signaling, and specifically deubiquitinates monoubiquitinated 

Smad4. 

USP10 G3BP, p53/ 

TP53, SNX3, 

NEMO, SirT6 

USP11 

USP12 

USP13/ 

ISOT3 

USP14 

USP15 

BRCA2, 

CHUK/IKKA, 

RANBP9/ 

RANBPM , 

PML, ALK5 

WDR48, 

PHLPP1, 

Notch 

PTEN, Stat1, 

Ubl4A 

FANCC, 

CXCR4, 

ERN1, REST, 

I-κB 

E6, ubH2B, 

TRIM25, 

KEAP1, 

REST, BRAP, 

TβR-I 


deubiquitinase superfamily. USP10 functions as an essential regulator of p53/TP53 

stability following DNA damage. 


deubiquitinase superfamily. USP11 aids in the regulation of pathways leading to 

NF-κB activation and also DNA repair after double-stranded DNA breaks. Depletion 

of USP11 causes inhibition of TGFβ-induced epithelial to mesenchymal transition. 


deubiquitinase superfamily. USP12 requires interaction with WDR48 for high 

deubiquitinase activity. WDR48, in complex with deubiquitinase USP12, suppresses 

Akt-dependent cell survival signaling by stabilizing PHLPP1. USP12 and its activator 

UAF1 deubiquitinate nonactivated Notch. 


deubiquitinase superfamily. USP13 is part of an autophagy regulatory loop involving 

the deubiquitination of USP10 that leads to regulation of p53 stability. USP13 is a 

tumor suppressing protein that functions through deubiquitylation and stabilization 

of PTEN. USP13 also modulates STAT1 and plays a role in host defense against 

viral infection. 


deubiquitinase superfamily. USP14 is one of three proteasome-associated deubiquitinases, 

along with POH and UCHL5. USP14 is thought to antagonize substrate 

degradation as a part of the proteasome. 


deubiquitinase superfamily. USP15 preferentially cleaves K48-linked polymers. 

USP15 deubiquitination protects APC and human papillomavirus type 16 protein E6 

target proteins against proteasomal degradation. USP15 is a critical regulator of the 

TRIM25- and RIG-I-mediated antiviral immune response. USP15 also regulates the 

TGF-β pathway and is a key factor in glioblastoma pathogenesis. 

USP16 H2A USP16 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP16 acts as a transcriptional co-activator by 

specifically targeting H2A for deubiquitination. USP16 deubiquitination of H2A 

is also required for entry into mitosis. 

USP17 

RIG-I, MDA- 

5, SDS3 

USP17 regulates virus-induced type I IFN signaling through deubiquitination of 

RIG-I and MDA5. USP17 specifically deubiquitinates Lys-63-linked ubiquitin chains 

from SDS3. 

USP18 TAK1, TAB1 USP18 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP18 catalyzes the removal of ISG15, an interferonregulated 

ubiquitin-like protein, which maintains the critical cellular balance of 

ISG15-conjugated proteins important for normal development and brain function. 

USP18 deubiquitinates the TAK1/TAB1 complex thus inhibiting NF-κB and NFAT 

activation during Th17 differentiation. 

USP19 RNF123 USP19 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP19 deubiquitinates target proteins involved in 

cell proliferation, myogenesis, regulation of hypoxia, and modulation of the ERAD 

protein degradation pathway. 

USP20 

VHL, DIO2, 

HIF1α 


deubiquitinase superfamily. USP20 cleaves K48- and K63-linked chains. USP20 

deubiquitinates β2-adrenergic receptor (ADRB2) as well as target proteins involved 

in thyroid hormone regulation and regulation of hypoxia. 

9628861, 

16520378, 

17711858, 

23154984, 

24378640, 

22253573 

16322459, 

18254724, 

19135894, 

23184937, 

23097624, 

24591637, 

23690623, 

22371489 

19246359 

11439350, 

18632802, 

19398555, 

24270572, 

24332849 

15314155, 

17897950, 

18408009, 

24487962, 

22773947 

19075014, 

24145035, 

22778262 

9841226, 

24270891, 

23940278, 

24424410 

18162577, 

19135427, 

19106094, 

23754622, 

23615914 

16005295, 

19576224, 

24526689, 

24399297, 

23727018, 

23708518, 

23105109, 

22344298 

10077596, 

17914355 

20368735, 

21239494 

10777664, 

23825189 

19465887 

12056827, 

12865408, 

15776016 





USP21 

USP22 

H2A, RIG-I, 

GATA-3 

ATXN7L3, 

NFATc2 


deubiquitinase superfamily. USP21 is also known as USP23. USP21 acts as a 

transcriptional co-activator by specifically targeting H2A for deubiquitination. 

USP21 is capable of removing the ubiquitin-like NEDD8 from NEDD8 conjugates. 

USP21 acts as a negative regulator in antiviral responses by binding and deubiquitinating 

RIG-I. USP21 also stabilizes the transcription factor GATA-3 by mediating its 

deubiquitination. 


deubiquitinase superfamily. USP22 deubiquitinates histones H2A and H2B as a 

component of the histone acetylation (HAT) complex SAGA. USP22 deubiquitinates 

specific targets required for transcription, nuclear receptor-mediated transactivation, 

and cell cycle progression. USP22 positively regulates NFATc2 through its 

deubiquitinase activity and promotes IL2 expression. 

10799498, 

24493797, 

23395819 

18206972, 

18206973, 

18469533, 

24561192 

USP23 H2A See USP21 10799498 

USP24 DDB-2 USP24 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. Mutations of the USP24 gene may correlate with risk 

of Parkinson’s disease. USP24 interacts with and regulates stability of the DNA 

damage specific protein, DDB-2. 

USP25 

ACTA1, 

MYBPC1, 

TRAF3, 

TRAF5, 

TRAF6 


deubiquitinase superfamily. USP25 cleaves both K48- and K63-linked chains. 

The USP25 muscle-specific isoform may have a role in the regulation of muscular 

differentiation and function. USP25 targets TRAF5 and TRAF6 for deubiquitination 

and thus negatively regulates IL-17-mediated signaling and inflammation. 

USP26 AR USP26 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP26 regulates the androgen receptor signaling 

pathway by targeting the androgen receptor for deubiquitination. 

USP27X 

USP27X is an X-linked member of the ubiquitin-specific processing protease (USP/ 

USB) deubiquitinase superfamily. 

USP28 

P53bp1, 

Chk2, LSD1 


deubiquitinase superfamily. USP28 can bind to and deubiquitinate several target 

proteins in the DNA damage pathway, resulting in their stability, including p53BP1 

and Chk2. USP28 also plays an important role in Myc related signaling by binding 

through FBW7α to Myc. USP28 stabilizes LSD1 via deubiquitination, and USP28 

overexpression has been linked to the upregulation of LSD1 upregulation in multiple 

cancer cell lines and breast tumor samples. 

USP29 Claspin USP29 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP29 helps regulate the ATR-Chk1 pathway and the 

control of DNA replication via Claspin deubiquitination. 

USP30 


deubiquitinase superfamily. USP30 may participate in the maintenance of mitochondrial 

morphology. 

USP31 


deubiquitinase superfamily. 

USP32 


deubiquitinase superfamily. USP32 is highly expressed in breast cancer cell lines 

and may be involved in tumorigenesis. 

USP33 

USP34 

USP35 

USP36 

USP37 

USP38 

ADRB2, 

CP110, 

RalB 

AXIN1, 

AXIN2 

RNA Polymerase 

I 

FZR1/CDH1, 

PLZF/RARα 


deubiquitinase superfamily. USP33 is involved in cellular migration, β2-adrenergic 

receptor/ADRB2 recycling, and G protein-coupled receptor (GPCR) signaling. In 

addition, USP33 regulates centrosome biogenesis by deubiquitinating CP110. 

USP33 also regulates the autophagy and innate immune response of RalB through 

deubiquitination of RalB. 


deubiquitinase superfamily. USP34 acts as an activator of the Wnt signaling 

pathway downstream of the β-catenin destruction complex by deubiquitinating and 

stabilizing AXIN1 and AXIN2. 




deubiquitinase superfamily. USP36 may play a role in the maintenance of stem 

cells and regulation of cellular differentiation. Furthermore, USP36 regulates rRNA 

production through control of RNA Polymerase I stability. 


deubiquitinase superfamily. USP37 antagonizes the anaphase-promoting complex 

(APC/C) during G1/S transition by mediating deubiquitination of cyclin A (CCNA1 and 

CCNA2), thereby promoting S phase entry. In addition, USP37 is involved in acute 

promyelocytic leukemia (APL) through regulating the stability of the oncogenic fusion 

protein PLZF/RARα. 


superfamily. USP38 is expressed in skeletal muscle and adrenal gland. 

16917932, 

23159851 

10612803, 

11597335, 

16501887, 

23674823, 

23042150 

20501646 

12838346 

17558397, 

16901780, 

24075993 

10958632, 

24632611 

18287522 

14715245 

12604796, 

20549504 

12865408, 

23486064, 

24056301 

21383061 

14715245 

22622177, 

22902402 

21596315, 

23208507 

19615732 


USP39 

USP39 is a member of the ubiquitin-specific processing protease (USP/USB) 11350945 

deubiquitinase superfamily. USP39 may play a role in mRNA splicing as a 

competitor of ubiquitin C-terminal hydrolases (UCHs). 

USP40 

USP40 may be a nonprotease homologue of the ubiquitin-specific processing 16917932 

protease (USP/USB) superfamily. 

USP41 



USP42 


deubiquitinase superfamily. USP42 may play a role in spermatogenesis. 

USP43 



USP44 Cdc20, 

Histone H2B 


deubiquitinase superfamily. USP44 regulates the cell cycle by deubiquitination of 

17443180, 

22681888 

CDC20, leading to stabilization of the MAD2L1-CDC20-APC/C ternary complex and 

avoidance of premature anaphase entry. USP44 modulates H2B ubiquitylation thus 

regulating stem cell differentiation. 

USP45 



14715245 

USP46 

USP47 

USP48 

GAD1/ 

GAD67, 

PHLPP 

POLB, 

CDC25A, 

katanin-p60 

TRAF2, 

RELA, NHE3 


deubiquitinase superfamily. USP46 requires interaction with WDR48 for high 

deubiquitinase activity. USP46 may act by mediating the deubiquitination of 

GAD1/GAD67. USP46 also regulates Akt signaling in colon cancer through 

control of PHLPP activation. 


superfamily. USP47 regulates base excision repair by deubiquitinating 

monoubiquitinated DNA polymerase β (POLB). USP47 may also regulate cell growth 

and survival by targeting CDC25A. USP47 promotes axonal growth of cultured 

rat hippocampal neurons through specifically deubiquitinating and stabilizing 

katanin-p60. 


deubiquitinase superfamily. USP48 may be involved in the regulation of NF-κB 

activation by the TNF receptor superfamily via its interactions with RelA and TRAF2. 

USP48 can also prevent NHE3 degradation by deubiquitination and thus helps 

regulate blood pressure and sodium balance. 

19075014, 

22391563 

19966869, 

23904609 

16214042, 

24308971 

USP49 Histone H2B USP49 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP49 specifically regulates Histone H2B by deubiquitination 

14715245, 

23824326 

and is required for efficient cotranscriptional splicing of exons. 

USP50 

USP50 is a nonprotease homologue of the ubiquitin-specific processing protease 14715245 

(USP/USB) superfamily. 

USP51 



14715245 

USP52 PAN3 USP52 is a member of the ubiquitin-specific processing protease (USP/USB) 

deubiquitinase superfamily. USP52 is a member of the Pan nuclease complex, 

which regulates mRNA stability. 

USP53 

USP53 is a nonprotease homologue of the ubiquitin-specific processing protease 


USP54 

USP54 is a nonprotease homologue of the ubiquitin-specific processing protease 


USPL1 

USPL1 is a nonprotease homologue of the ubiquitin-specific processing protease 


USPL2/ 

CYLD 

NF-κB, 

HDAC6, 

RIP1 

CYLD deubiquitinase regulates inflammation and cell proliferation by down regulating 

NF-κB signaling through removal of ubiquitin chains from several NF-κB pathway 

proteins. CYLD is a negative regulator of proximal events in Wnt/β-catenin signaling 

and is a critical regulator of natural killer T cell development. In selenite-treated 

colorectal cancer cells, CYLD regulates RIP1 deubiquitination and triggers apoptosis. 

VCPIP1 VCP VCPIP1 (valosin containing protein p97/p47 complex-interacting protein) is a 

member of the deubiquitinating enzyme ovarian tumor domain (OTU) superfamily. 

VCPIP1 is necessary for VCP-mediated reassembly of Golgi stacks after mitosis. 

14583602, 

16284618 

14715245 

14715245 

12917689, 

12917690, 

24577083 

15037600 



05 


Cellular Metabolism 

Energy is indispensible for all forms of life, and glucose is the primary energy source for most cells of 

the body. Glucose metabolism is central to cell proliferation, growth, survival, and tumor progression. 

Insulin Signaling and Glucose Transport 

The maintenance of glucose homeostasis is an essential physiological process that is regulated 

by hormones. An elevation in blood glucose levels during feeding stimulates insulin release from 

pancreatic β cells through a glucose-sensing pathway. Insulin stimulates glucose uptake from blood 

into skeletal muscle, cardiac muscle, and adipose tissue through a signaling cascade mediated by the 

insulin receptor (IR). Insulin binding to the IR results in activation of the insulin receptor substrate (IRS) 

proteins and subsequent signaling to the PI3K/Akt and Erk1/2 pathways, resulting in translocation of 

Glut4 from vesicles to the plasma membrane, glucose uptake, cell proliferation, and survival. Glut2, 

on the other hand, mediates glucose transport across liver cell membrane where glucose is converted 

to glycogen. When blood glucose levels are high, insulin suppresses glucose production in the liver. 

When blood glucose levels are low, liver cells degrade glycogen to glucose and/or synthesize glucose 

from noncarbohydrate substrates to release it into the blood. Aberrant insulin signaling can result in 

diabetes, obesity, atherosclerosis, and neurodegenerative disease. 

The insulin-like growth factor-I (IGF-I) receptor is similar in structure to the IR and can exist either as a 

homodimer or as a heterodimer with the IR. IGF-I receptor uses a similar signaling cascade as the IR to 

promote cell proliferation and survival. 

AMPK Signaling 

During conditions of glucose deprivation when cellular ATP levels fall, the serine/threonine kinase 

AMPK becomes active. AMPK is an energy sensor that is activated by an elevated AMP:ADP/ATP ratio 

due to cellular and environmental stress, such as low glucose, heat shock, hypoxia, or ischemia. AMPK 

activation positively regulates signaling pathways that replenish cellular ATP supplies. For example, 

activation of AMPK enhances both the transcription and translocation of Glut4, resulting in an increase 

in insulin-stimulated glucose uptake. It also stimulates catabolic processes such as fatty acid oxidation 

and glycolysis via inhibition of ACC and activation of PFK2. AMPK also activates ULK1 to trigger 

autophagy, which recycles cellular components for energy production. AMPK negatively regulates 

several proteins central to ATP-consuming processes such as mTORC2, glycogen synthase, SREBP-1, 

and TSC2, resulting in the downregulation or inhibition of gluconeogenesis and of glycogen, lipid, and 

protein synthesis. 

ACC, a key enzyme in the biosynthesis and 

oxidation of fatty acids, is commonly phosphorylated 

at Ser79 in breast carcinoma. 

Phospho-Acetyl-CoA Carboxylase (Ser79) (D7D11) Rabbit mAb #11818: 

IHC analysis of paraffin-embedded human breast carcinoma using #11818. 

chapter 05: cellular metabolism 

Oligomycin treatment 


of AMPKα at 

Thr172. 

kDa 

100 

80 

60 

50 

40 

30 

100 

80 

60 

50 

40 

30 

– + 

Phospho- 

AMPKα 

(Thr172) 

AMPKα 

Oligomycin 

Phospho-AMPKα (Thr172) (40H9) 


extracts from C2C12 cells, untreated 

or oligomycin-treated (0.5 µM), using 

#2535 (upper) or AMPKα Antibody 

#2532 (lower). 


for IGF-I/II Signaling 

#8917 Human Insulin-like Growth 

Factor I (hIGF-I) 

#5238 Human Insulin-like Growth 

Factor II (hIGF-II) 

#9897 Mouse Insulin-like Growth 

Factor I (mIGF-I) 

Insulin expression in rat pancreas 

Stimulation of cells with IGF-I or insulin 

results in phosphorylation of IGF-I receptor 

β at Tyr1135/1136 and insulin receptor β at 

Tyr1150/1151. 

Phospho-IGF-I-Receptor β (Tyr1135/1136)/Insulin Receptor β (Tyr1150/1151) 

(19H7) Rabbit mAb #3024: WB analysis of extracts from untreated and IGF-treated 

HeLa cells and untreated and insulin-treated H-4-II-E cells using #3024. 

Insulin (C27C9) Rabbit mAb #3014: Confocal IF analysis of rat pancreas 

using #3014 (green). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). 

kDa 

140 

100 

80 

60 

50 

40 

30 

HeLa 

H-4-II-E 

Phospho-IGF-I 

Receptor β/Insulin 

Receptor β 

Cancer Metabolism 

The role of metabolism in cancer has been a focus of extensive research. In the 1920s, Otto Warburg 

observed that tumor cells produce energy by undergoing high rates of glycolysis and lactate fermentation 

in the cytosol in the presence of oxygen, a condition usually found only in the oxygen-deprived 

state. In contrast, normal cells typically have lower rates of glycolysis followed by oxidation of pyruvate 

in mitochondria. This cancer-associated phenomenon was later termed the Warburg Effect. Several 

signaling pathways such as Akt, Erk1/2, and AMPK converge on the key glycolytic enzymes phosphofructokinase 

(PFK) and pyruvate kinase M2 isoform (PKM2) to regulate ATP synthesis. The transcription 

factors c-Myc and p53 regulate glutamine metabolism, lipid metabolism, and the pentose phosphate 

pathway, creating the cellular components (nucleotides, lipids, proteins, Krebs Cycle intermediates) 

necessary for supporting and sustaining rapid tumor cell growth. 

Hexokinase II, an enzyme that catalyzes the first 

step in glycolysis, is expressed in lung carcinoma. 

Hexokinase II (C64G5) Rabbit mAb #2867: IHC analysis of paraffin-embedded human 

lung carcinoma using #2867. 

Glycolytic enzyme 

PKM2 is expressed in 

cancer cells. 

PKM2 (D78A4) XP ® Rabbit mAb 


human lung carcinoma using #4053. 

20 

– + – – 

– – – + 

IGF 

Insulin 

Chemical Modulators 

for AMPK Signaling 

Insulin stimulation results in phosphorylation 

of AS160 at Thr642, a key event in insulinstimulated 

glucose transport. 

Phospho-AS160 (Thr642) (D27E6) Rabbit mAb #8881: WB analysis of extracts 

from serum starved HeLa cells, untreated (-) or insulin-treated (150 nM, 15 min), using 

#8881 (upper) or AS160 (C69A7) Rabbit mAb #2670 (lower). The phospho-specificity 

of the antibody is verified by λ phosphatase treatment. 

kDa 

200 

140 

100 

80 

60 

50 

200 

140 

100 

80 

60 

50 

– + 

– – 

– + Insulin 

+ + λ phosphatase 

Phospho-AS160 

(Thr642) 

AS160 


Burkewitz, K., Zhang, Y., and Mair, W.B. (2014) Cell Metab. 20, 10−25. • Guo, S. (2014) J. Endocrinol. 220, T1−T23. • Iqbal, 

M.A., Gupta, V., Gopinath, P., et al. (2014) FEBS Lett. 588, 2685–2692. • Metallo, C.M. and Vander Heiden, M.G. (2013) Mol. 

Cell 49, 388−398. • Mouchiroud, L., Eichner, L.J., Shaw, R.J., et al. (2014) Cell Metab. 20, 26−40. • Richter, E.A. and 

Hargreaves, M. (2013) Physiol. Rev. 93, 993−1017. • Ruderman, N.B., Carling, D., Prentki, M., et al. (2013) J. Clin. Invest. 

123, 2764−2772. • Yang, W. and Lu, Z. (2013) Cancer Lett. 339, 153−158. • Zhang, C., Liu, J., Liang, Y., et al. (2013) Nat. 

Commun. 4, 2935. 

#9944 AICAR 

AMP analog that 

activates AMPK 

#9996 Oligomycin 

ATP synthase inhibitor; inhibits 

oxidative phosphorylation; 

Activates AMPK 


www.cellsignal.com/cstcellularmetabolism 141



nodes within cellular metabolism signaling 


in cellular metabolism research. Primary 











Cytoplasmic protein 

IDH1 catalyzes the 

oxidative decarboxylation 

of isocitrate to 

α-ketoglutarate and 

functions as a tumor 

suppressor. 

IDH1 (D2H1) Rabbit mAb #8137: 

Confocal IF analysis of 293T cells using 



Commonly Studied Cellular Metabolism Targets Target M P E S C 

Phospho-IGF-I Receptor β (Tyr980) • 

Target M P E S C Target M P E S C 

Phospho-IGF-I Receptor β (Tyr1131)/ • • 

4F2hc/CD98 

• 

C1QBP 

• • 

Insulin Receptor β (Tyr1146) 

ABCC4 

• • 

CA2 

• 


ABCG2 

• 

CA9 

• 

Phospho-IGF-I Receptor β • • 

ACAD9 

• 

CA12 

• 

(Tyr1135/1136)/Insulin Receptor β 

ACAD10 

• CAD 

• 

(Tyr1150/1151) 

ACAT2 

• 

Phospho-CAD (Ser1859) • 


Acetyl-CoA Carboxylase • • • • Catalase 

• 

IGFBP-2 

• 

Phospho-Acetyl-CoA Carboxylase • • • • CLIC4 

• 

IGFBP-3 

• 

(Ser79) 

COX IV • • • 

Insulin Receptor β • • • 

Acetyl-CoA Carboxylase 1 • • CPT1A 

• 

Phospho-Insulin Receptor β (Tyr1146) • 

Acetyl-CoA Carboxylase 2 • 

CRABP1 

• 

Phospho-Insulin Receptor β • 

AceCS1 

• 

(Tyr1150/1151) 

CCTα 

• • 

ACO2 

• • 

Phospho-Insulin Receptor β (Tyr1345) 

CTR1/SLC31A1 

• 

• • 

ACSL1 

• • 

Phospho-Insulin Receptor β (Tyr1361) 

CYP11A1 

• 

• 

ADH1 

• 

Insulin 

CYP3A4 

• 

• • • 

Adiponectin 

• 

C-Peptide 

DHCR24/Seladin-1 • 

• • 

AKR1C2 

• 

IRAP 

DLST 

• • 

• • 

ALDH1A1 

• 

IRS-1 

DPYD 

• 

• • • • 

Aldolase A • • • 

Phospho-IRS-1 (panTyr) 

Enolase-1 

• • 

• 

AMACR 

• 

Phospho-IRS-1 (Ser302) 

Enolase-2 

• • 

• • • 

AMPKα • • • • 


ENPP1 

• • 

• • 

Phospho-AMPKα (Thr172) • • • • 


FAAH1 

• • 

• 

AMPK-α1 

• 

Phospho-IRS-1 (Ser332/Ser336) 

FABP1 

• 

• 

Phospho-AMPKα1 (Ser485) • • 


FABP4 

• • 

• • • 

Phospho-AMPKα1 (Ser485)/AMPKα2 • 

Phospho-IRS-1 (Ser636/Ser639) 

Fatty Acid Synthase • • • • 

• 

(Ser491) 


FTH1 

• 

• 

AMPK-α2 • • 

Phospho-IRS-1 (Tyr895) 

Fetuin A 

• 

• 

AMPK-β1 

• • 


FHIT 

• 

• 

Phospho-AMPK-β1 (Ser108) • 

Phospho-IRS-1 (Tyr1222) 

Phospho-FHIT (Tyr114) • 

• 

Phospho-AMPK-β1 (Ser182) • 

IRS-2 

FoxA2/HNF3β 

• • 

• • 

AMPK-β1/β2 

• 

Phospho-IRS-2 (Tyr) 

FoxC2 

• 

• 

AMPK-β2 

• 

LAT1 

Fumarase 

• 

• 

AMPK-γ1 

• 

LDH-A 

Glucose-6-Phosphate Dehydrogenase • • 

• • 

AMPK-γ2 

• 

LDH-A/LDH-C 

GAPDH • • 

• 

AMPK-γ3 

• 

Phospho-LDH-A (Tyr10) 

GATA-2 

• 

• 

Amylase 

• • 

Lipin 1 

GFAT1 

• • 

• 

ANT2/SLC25A5 

• 

LKB1 

GFAT2 

• 

• 

AQP2 

• 

Phospho-LKB1 (Thr189) 

GLDC 

• 

• 

Arginase-1 

• 

Phospho-LKB1 (Ser334) 

Glucagon • • • 

• 

ARK5 

• 

Phospho-LKB1 (Ser428) 

Glut4 

• • 

• 

AS160 

• • 

LXR-β 

Glycogen Synthase • • 

• 

Phospho-AS160 (Ser318) • 

Malic Enzyme (Mitochondrial) 

Phospho-Glycogen Synthase (Ser641) • 

• 

Phospho-AS160 (Ser588) • 

MDH2 

GPX1 

• • 

• • 

Phospho-AS160 (Thr642) • • 

MDR1/ABCB1 

GUCY1A2 

• 

• 

ASCT2 

• • 

Mitofusin-1 

hERG1α 

• 

• 

ATF-4 

• 

Mitofusin-2 

Hexokinase I 

• • 

• 

ATGL 

• • 

MO25α/CAB39 

Hexokinase II 

• • 

• 

ATP Citrate Lyase 

• • 

MRP1/ABCC1 

HMOX2/HO-2 

• 

• 

Phospho-ATP Citrate Lyase (Ser455) • 

MRP2 

HNF-1α 

• 

• • 

ATPIF1 

• • 

MRP3/ABCC3 

HNF4α 

• • 

• 

BCAT1 

• 

MRP4/ABCC4 

HO-1 

• • 

• 

BCAT2 

• 

MTAP 

HSL 

• 

• • 

C/EBP-α 

• • 

NME1/NDKA 

Phospho-HSL (Ser563) • 

• • 

Phospho-C/EBPα (Ser21) • 

NPC1L1 


• 

Phospho-C/EBPα (Thr222/226) • 

NQO1 


• 

C/EBP-β 

• 

NRF1 

IDH1 

• • 

• 

Phospho-C/EBPβ (Ser105) • 

OGDH 

IDH2 

• 

• 

Phospho-C/EBPβ (Thr235) • 

O-GlcNAc 

IGF-I Receptor 

• 

• • 

C/EBP-δ 

• 

OGT 

IGF-I Receptor β • • • • 

• 

PANK2 

• 



PANK4 

• 

PASK 

• 

PCK1 

• 

PCK2 

• • 

Pyruvate Dehydrogenase • • 

PDHK1 

• 

Pdx1 

• 

Perilipin 

• • 

PFKFB2 

• 

Phospho-PFKFB2 (Ser483) • 

PFKFB3 

• 

PFKL 

• 

PFKP 

• • 

PGAM1 

• 

PGC1α 

• 

PGD 

• 

PGRMC1 

• • 

PHGDH 

• 

PiT1/SLC20A1 

• 

PKM1 

• 

PKM1/2 

• • 

PKM2 • • • 

Phospho-PKM2 (Tyr105) • 

PLA2G1B 

• 

Prdx1 

• 

PTP1B 

• 

SCAP 

• 

SCD1 

• • 

SDHA 

• • 

SGLT1 

• 

SHMT1 

• 

SHMT2 

• 

SIK2 

• 

SLC4A4/NBC1 

• 

SLC7A11/xCT 

• 

SNARK/NUAK2 

• 

SOD2 

• 

Phospho-SREBP-1c (Ser372) • 

StAR 

• 

Succinyl-CoA Synthetase • • 

Synip 

• 

TBC1D1 

• 

Phospho-TBC1D1 (Thr590) • 

Phospho-TBC1D1 (Ser660) • 

Phospho-TBC1D1 (Ser700) • 

Thioredoxin 1 

• • 

Thioredoxin 2 

• 

Thymidine Kinase 1 

• 

Thymidylate Synthase • • 

TORC1/CRTC1 

• • 

Phospho-TORC1/CRTC1 (Ser151) • 

TORC2/CRTC2 

• • 

TORC3/CRTC3 

• • 

Transketolase 

• 

TRXR1 

• 

Tug 

• 

TRXR2/TXNRD2 

• 

Tyrosinase 

• 

UGT 

• 

VMS1 

• 


220 


CST antibodies for Phospho-AMPK 

(Thr172) have been cited over 220 times 




Murthy, A. et al. (2014) A Crohn’s 

disease variant in Atg16l1 enhances 

its degradation by caspase 3. Nature 

506, 456−462. 

Sakamaki, J. et al. ((2014) Role of the 

SIK2-p35-PJA2 complex in pancreatic 

beta-cell functional compensation. 

Nature Cell Biol. 16, 234−244. 

Liu, P.P. et al. (2014) Metabolic 

regulation of cancer cell side population 

by glucose through activation of 

the Akt pathway. Cell Death Differ. 21, 

124−135. 

Takayama, H. et al. (2014) Metformin 

suppresses expression of the selenoprotein 

P gene via an AMP-activated 

kinase (AMPK)/FoxO3a pathway in 

H4IIEC3 hepatocytes. J. Biol. Chem. 

289, 335−345. 

Wu, Y. et al. (2014) Phosphorylation of 

p53 by TAF1 inactivates p53-dependent 

transcription in the DNA damage 

response. Mol. Cell 53, 63−74. 

Wang, L. et al. (2014) Pten deletion 

in RIP-Cre neurons protects against 

type 2 diabetes by activating the 

anti-inflammatory reflex. Nat. Med. 

20, 484−492. 

Jang, Y.H. et al. (2014) Phospholipase 

D-mediated autophagic regulation is 

a potential target for cancer therapy. 

Cell Death Differ. 21, 533−546. 

Thompson, A.M. et al. (2014) 

Resveratrol induces vascular smooth 

muscle cell differentiation through 

stimulation of SirT1 and AMPK. PLoS 

One 9, e85495. 

Cheng, H. et al. (2014) A genetic 

mouse model of invasive endometrial 

cancer driven by concurrent loss of 

Pten and Lkb1 Is highly responsive 

to mTOR inhibition. Cancer Res. 74, 

15−23. 

Demir, U. et al. (2014) Metformin 

anti-tumor effect via disruption of the 

MID1 translational regulator complex 

and AR downregulation in prostate 

cancer cells. BMC Cancer 14, 52. 

Wang, W. et al. (2014) Human T-cell 

leukemia virus type 1 Tax-deregulated 

autophagy pathway and c-FLIP 

expression contribute to resistance 

against death receptor-mediated 

apoptosis. J. Virol. 88, 2786−2798. 

Spruiell, K. et al. (2014) Role of 

pregnane X receptor in obesity and 

glucose homeostasis in male mice. 

J. Biol. Chem. 289, 3244−3261. 

www.cellsignal.com/cstcellularmetabolism 143


Insulin Receptor Signaling 

Glucose 

GLUT4 

PDK1 

Bad 

SGK 

Apoptosis 

ENaC 

Sodium 

Transport 

Cytoplasm 

Nucleus 

GLUT4 

Translocation 

GLUT4 

vesicle 

GLUT4 

Exocytosis 

PP1 

mTORC1 

GβL Raptor 

mTOR 

DEPTOR 

FoxO3 

SNARE 

Complex 

Synip 

TC10 

CIP4/2 

GSK-3 

SGK 

Crkll 

PP2A 

14-3-3 

AS160 

TBC1D1 

14-3-3 

GS 

Glycogen 

Synthesis 

Transcription 

CAP 

C3G 

Rac1 

ATP-citrate 

lyase 

FoxO4 

Flotillin 

Cav 

SREBP 

Fatty Acid 

Synthesis 

mTORC2 

Sin1 PRR5 

Rictor GβL 

mTOR 

DEPTOR 

Cbl 

APS 

EHD1 

EHBP1 

PKCλ/ζ 

Lipin1 

Akt2 

FoxO1 

PDK1 

PTEN 

SHIP 

PRAS40 

Apoptosis, 

Autophagy, 

Glucose and 

Lipid Metabolism 

4E-BP1 

PIP 3 

Akt 

TSC2 

TSC1 

Rheb 

p85 

PI3K 

p110 


Gab1 

SHP-2 

IRS 

Shc 

GRB2 

IRS-1 

eIF4E 

HSL 

Protein Synthesis, 

Growth, and SREBP-1 

Proliferation 

Lipolysis 

Lipin1 

Akt2 

mTORC2 

mTORC1 

Fatty Acid 

and Cholesterol 

Synthesis 

PKCλ/ζ 

LKB1 

AMPK 

SREBP 

p70 

S6K 

Erk1/2 

LXRα 

PDE3B 

[cAMP] 

PKA 

USF 

Insulin Receptor 

PKCθ 

FFA 

NO 

SIK2 

SOCS3 

Nck 

Crkll 

Fyn 

IKK 

ROS 

CBP/p300 

PTP1B 

iNOS 

Jnk 

Torc2 

Disruption of 

CBP/Torc2/CREB 

Complex 


TNFR1 

TNF 

GRB10 

Degradation 

SOS 

Ras 

c-Raf 

MEK1/2 

Erk1/2 

Erk1/2 

Growth 

Insulin is the major hormone controlling critical energy functions such as glucose and lipid metabolism. Insulin activates the insulin receptor tyrosine kinase (IR), which 

phosphorylates and recruits different substrate adaptors such as the IRS family of proteins. Tyrosine phosphorylated IRS then displays binding sites for numerous signaling 

partners. Among them, PI3K has a major role in insulin function, mainly via the activation of the Akt/PKB and the PKCζ cascades. Activated Akt induces glycogen synthesis 

through inhibition of GSK-3; protein synthesis via mTOR and downstream elements; and cell survival through inhibition of several pro-apoptotic agents (Bad, FoxO transcription 

factors, GSK-3, and MST1). Akt phosphorylates and directly inhibits FoxO transcription factors, which also regulate metabolism and autophagy. Inversely, AMPK is known to 

directly regulate FoxO3 and activate transcriptional activity. Insulin signaling also has growth and mitogenic effects, which are mostly mediated by the Akt cascade as well as 

by activation of the Ras/MAPK pathway. The insulin signaling pathway inhibits autophagy via the ULK1 kinase, which is inhibited by Akt and mTORC1, and activated by AMPK. 

Insulin stimulates glucose uptake in muscle and adipocytes via translocation of GLUT4 vesicles to the plasma membrane. GLUT4 translocation involves the PI3K/Akt pathway 

and IR-mediated phosphorylation of CAP, and formation of the CAP:CBL:CRKII complex. In addition, insulin signaling inhibits gluconeogenesis in the liver, through disruption 

of CREB/CBP/mTORC2 binding. Insulin signaling induces fatty acid and cholesterol synthesis via the regulation of SREBP transcription factors. Insulin signaling also promotes 

fatty acid synthesis through activation of USF1 and LXR. A negative feedback signal emanating from Akt/PKB, PKCζ, p70 S6K, and the MAPK cascades results in serine 

phosphorylation and inactivation of IRS signaling. 


Altarejos, J.Y. and Montminy, M. (2011) Nat. Rev. Mol. Cell Biol. 12, 141–151. • Cheng, Z., Tseng, Y., and White, M.F. (2010) Trends Endocrinol. Metab. 21, 589–958. 

• Fritsche, L., Weigert, C., Häring, H.U., and Lehmann, R. (2008) Curr. Med. Chem. 15, 1316–1329. • Guo, S. (2014) J. Endocrinol. 220,T1–T23. • Rowland, A.F., 

Fazakerley, D.J., and James, D.E. (2011) Traffic 12, 672–681. • Siddle, K. (2011) J. Mol. Endocrinol. 47, R1–R10. • Shao, W. and Espenshade, P.J. (2012) Cell Metab. 

16, 414–419. • Wong, R.H. and Sul, H.S. (2010) Curr. Opin. Pharmacol. 10, 684–691. 

© 2003–2015 Cell Signaling Technology, Inc. • We would like to thank Ashley Webb and Prof. Anne Brunet Stanford University, Palo Alto, CA for reviewing this diagram. 


Pentose 

Phosphate 

Shunt 

NADPH 

Ribulose-5P 

Nucleotide 

Synthesis 

NADPH 

Folate 

Metabolism 

6-P- 

NADP 

Gluconate 

Glycine 

NADP 

Macropinocytosis and 

other scavenging pathways 

Malic Enzyme 

Akt 

Amino Acids 

and Lipids 

Serine 

Malate 

Hexokinase 

NADP 

PHGDH 

OMM Stability 

Inhibition Cytochrome C Release 

Inhibition of Apoptosis 

Bax 

NADPH 

NADPH 

6-P- 

Gluconolactone 

p53 

ANT 

VDAC 

VDAC 

Lactate 

ANT 

TIGAR 

HIF-1α 

c-Myc 

NADP 

Malate 

Glucose 

Transporters 

Hexokinase 

Glucose-6-P 

Fructose-6-P 

PFK 

Fructose 

Bisphosphate 

3-Phosphoglycerate 

p53 

Glycolysis 

Glucose 

PEP 

PKM2 

Pyruvate 

IDH2 

Ras 

Citrate 

UCP2 

Ras 

LDHA 

Pyruvate PDHK 

Dehydrogenase 

Pyruvate 

Acetyl-CoA 

Oxaloacetate 

Krebs 

Cycle 

α-Ketoglutarate 

AMPK 

c-Myc 

c-Myc 

ULK1 

NADP 

Glutaminase 

NADPH 

Glutamine Transporters 

Glutamine 

Glutaminolysis 

LKB1 




PI3K 

Akt 

mTOR 

Autophagy 

β-Oxidation 

Citrate 

ACL 

Acetyl-CoA 

IDH1 ACC 

Epigenetic 

Regulation 

Ras 

Amino Acids 

SREBP 

HIF-1α 

Cell Proliferation 

MEK1/2 

Erk1/2 

c-Myc 

Fatty Acid Oxidation 

Fatty Acids 

Acetate 

ACSS2 

AMPK 

Fatty Acid/ 

Lipid Synthesis 

Cancer cells rely on a variety of metabolic fuels, and the specific nutrients used are impacted by both the genetic and environmental context of the cancer cell. 

Most mammalian cells use glucose as a fuel source. Glucose is metabolized by glycolysis in a multistep set of reactions resulting in the creation of pyruvate. In typical cells 

under normal oxygen levels, much of this pyruvate enters the mitochondria where it is oxidized by the Krebs Cycle to generate ATP to meet the cell’s energy demands. However, 

in cancer cells or other highly proliferative cell types, much of the pyruvate from glycolysis is directed away from the mitochondria to create lactate through the action of 

lactate dehydrogenase (LDH/LDHA)—a process typically reserved for the low oxygen state. Lactate production in the presence of oxygen is termed “aerobic glycolysis” or the 

Warburg Effect. 

Cancer cells frequently use glutamine as another fuel source, which enters the mitochondria and can be used to replenish Krebs Cycle intermediates or to produce more pyruvate 

through the action of malic enzyme. Highly proliferative cells need to produce excess lipid, nucleotide, and amino acids for the creation of new biomass. Excess glucose is 

diverted through the pentose phosphate shunt (PPS) and serine/glycine biosynthesis pathway to create nucleotides. Fatty acids are critical for new membrane production and 

are synthesized from citrate in the cytosol by ATP-citrate lyase (ACL) to generate acetyl-CoA. Acetate can also be a source of carbon for acetyl-CoA production when available. 

De novo lipid synthesis requires NADPH reducing equivalents, which can be generated through the actions of malic enzyme, IDH1, and also from multiple steps within the PPS 

pathway and serine/glycine metabolism. These reducing equivalents are also part of the defense against the increased levels of reactive oxygen species that are characteristic 

of cancer cells. There is also evidence that some cancer cells can scavenge extracellular protein, amino acids, and lipids. Macropinocytosis, a process that allows bulk uptake 

of extracellular material that can be delivered to the lysosome, is one way the cells can catabolize extracellular material and provide nutrients for cell metabolism. These 

nutrients can generate ATP or NADPH, or contribute directly to biomass. 

Several signaling pathways contribute to the Warburg Effect and other metabolic phenotypes of cancer cells. Growth factor stimulation results in signaling through RTKs to 

activate PI3K/Akt and Ras. Akt promotes glucose transporter activity and stimulates glycolysis through activation of several glycolytic enzymes including hexokinase and phosphofructokinase 

(PFK). Akt phosphorylation of apoptotic proteins such as Bax makes cancer cells resistant to apoptosis and helps stabilize the outer mitochondrial membrane 

(OMM) by promoting attachment of mitochondrial hexokinase (mtHK) to the VDAC channel complex. RTK signaling to c-Myc results in transcriptional activation of numerous 

genes involved in glycolysis and lactate production. The p53 oncogene transactivates TP-53-induced Glycolysis and Apoptosis Regulator (TIGAR) and results in increased 

NADPH production by PPS. Signals impacting levels of hypoxia inducible factor (HIF) can increase expression of enzymes such as LDHA to promote lactate production, as 

well as pyruvate dehydrogenase kinase to inhibit the action of pyruvate dehydrogenase and limit entry of pyruvate into the Krebs Cycle. There is also increasing evidence that 

availability of metabolic substrates can influence gene expression by affecting epigenetic marks on histones and DNA. 


Dang, C.V., Le, A., and Gao, P. (2009) Clin. Cancer Res. 15, 6479–6483. • Gogvadze, V., Zhivotovsky, B., and Orrenius, S. (2010) Mol. Aspects Med. 31, 60–74. • 

Hensley, C.T., Wasti, A.T., and DeBerardinis, R.J. (2013) J. Clin. Invest. 123, 3678–3684. • Kaelin, W.G. Jr. and McKnight, S.L. (2013) Cell 153, 56–69. • Lunt, S.Y. and 

Vander Heiden, M.G. (2011) Annu. Rev. Cell Dev. Biol. 27, 441–464. • Samudio, I., Fiegl, M., and Andreeff, M. (2009) Cancer Res. 69, 2163–2166. • Tennant, D.A., 

Durán, R.V., and Gottlieb, E. (2010) Nat. Rev. Cancer 10, 267–277. • White, E. (2013) Genes Dev. 27, 2065–2071. • Vander Heiden, M.G., Cantley, L.C., and Thompson, 

C.B. (2009) Science 324, 1029–1033. 

© 2010–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Matthew G. Vander Heiden, Massachusetts Institute of Technology, Cambridge, MA for reviewing this diagram. 




AMPK Signaling 

Acute Regulation of Metabolism 

Glucose 

Glut4 

Glut 

Trafficking 

Glycogen 

Synthesis 

Glycolysis 

GS 

PFKFB3 

Sterol/Isoprenoid 

Synthesis 

Fatty Acid 

Oxidation 

Glut1 

Leptin 

Glut4 

vesicle 

HMG-CoA 

Reductase 

CPT1 

ATGL 

Lipolysis 

Glut1 

vesicle 

Glut 

Translocation 

HSL 

Adiponectin 

Receptor 

Malonyl 

CoA 

Tau 

CLIP170 

Microtubules 

Cytoskeletal Signaling 

TXNIP 

TBC1D1 

α-Adrenergic 

Receptor 

PLCβ Gα q 

[cAMP] 

AMP + ADP 

ATP 

PKA 

LKB1 

β γ 

AMPKα 

p300 

SirT1 

[Ca 2+ ] 

CaMKK 

ACC 

Myosin 

Light 

Chain2 

Actin 

Dynamics 

Brain 

MYPT1 

MBS85 

CRY1 

STRAD 

MO25α 

HNF4α 

HDAC4 

HDAC5 

HDAC7 TORC2/ 

CRTC2 

FoxO 

Low Glucose, 

Hypoxia, Ischemia, 

Heat Shock 

AICAR 

Exercise 

Histamine 

Thrombin 

CREB 

PGC1α 


Ras 

FIP200 ATG13 

SREBP-1 

B-Raf 

Erk 

p90RSK 

ULK1 

Other RTKs 

PI3K 

Akt 

TSC2 

TSC1 

Raptor 

mTOR GβL 

4E-BP1 

p70 

S6K 

Autophagy 

Beclin 

PI3K Atg14 

Class III 

Fatty Acid 

Synthase 

Lipogenesis 

Transcriptional Control of Metabolism 

Insulin 

Receptor 

AMP-activated protein kinase (AMPK) plays a key role as a master regulator of cellular energy homeostasis. The kinase is activated in response to stresses that deplete 

cellular ATP supplies such as low glucose, hypoxia, ischemia, and heat shock. It exists as a heterotrimeric complex composed of a catalytic α subunit and regulatory β and 

γ subunits. Binding of AMP to the γ subunit allosterically activates the complex, making it a more attractive substrate for phosphorylation on Thr172 in the activation loop of 

the α subunit by its major upstream AMPK kinase, LKB1. AMPK can also be directly phosphorylated on Thr172 by CaMKK2 in response to changes in intracellular calcium as 

occurs following stimulation by metabolic hormones including adiponectin and leptin. 

As a cellular energy sensor responding to low ATP levels, AMPK activation positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid 

oxidation and autophagy. AMPK negatively regulates ATP-consuming biosynthetic processes including gluconeogenesis, lipid and protein synthesis. AMPK accomplishes 

this through direct phosphorylation of a number of enzymes directly involved in these processes as well as through transcriptional control of metabolism by phosphorylating 

transcription factors, co-activators, and co-repressors. 

Due to its role as a central regulator of both lipid and glucose metabolism, AMPK is considered to be a potential therapeutic target for the treatment of type II diabetes mellitus, 

obesity, and cancer. AMPK has also been implicated in a number of species as a critical modulator of aging through its interactions with mTOR and sirtuins. 


Cantó, C. and Auwerx, J. (2010) Cell. Mol. Life Sci. 67, 3407–3423. • Carling, D., Mayer, F.V., and Sanders, M.J., Gamblin SJ (2011) Nat. Chem. Biol. 512–518. • Hardie, 

D.G. (2011) Genes Dev. 25, 1895–1908. • Hardie, D.G., Ross, F.A., and Hawley, S.A. (2012) Chem. Biol. 19, 1222–1236. • Mihaylova, M.M. and Shaw, R.J. (2011) Nat. 

Cell Biol. 13, 1016–1023. • Steinberg, G.R. and Kemp, B.E. (2009) Physiol. Rev. 89, 1025–1078. • Zhang, B.B., Zhou, G., and Li, C. (2009) Cell Metab. 9, 407–416. 

Insulin 

Rheb 

Protein Metabolism and Autophagy 

Substrate 

ACC1 

AMPKA 

Isoform 

AMPKA1 

AMPKA2 



human S80 S80 LHIRssMsGLHLVkQ 17276402 

19176702 

18303014 

15371448 

AMPKA2 mouse S79 S80 FHMRSSMsGLHLVKQ 15866171 

AMPKA1 rat 

AMPKA2 

S79 

S1200 

S1215 

S80 

S1201 

S1216 

FHMRSsMsGLHLVKQ 

IPTLNRMsFASNLNH 

YGMTHVAsVSDVLLD 

12015362 

7915280 

1688796 

2900138 

1967580 



Acetyl-CoA carboxylase (ACC) catalyzes 

the carboxylation of acetyl-CoA to malonyl- 

CoA in the biosynthesis and oxidation 

of fatty acids. Phosphorylation by AMPK 

inhibits the enzymatic activity of ACC. 

ACC2 AMPKA1 human S222 S222 PTMRPSMsGLHLVKR 17276402 Acetyl-CoA carboxylase (ACC) catalyzes 

the carboxylation of acetyl-CoA to malonyl- 

CoA in the biosynthesis and oxidation 

of fatty acids. Phosphorylation by AMPK 

inhibits the enzymatic activity of ACC. 

AMPKA1 AMPKA1 human S360 

S486 

S494 

S496 

T183 

T388 

AMPKA1 rat S496 

T183 

T269 

AMPKB1 AMPKA1 human S24 

S108 

S174 

S177 

T80 

T158 

AMPKA1 rat S24 

S25 

S96 

S101 

S108 

S182 

S360 

S486 

S494 

S496 

T183 

T388 

S496 

T183 

T269 

S24 

S108 

S174 

S177 

T80 

T158 

S24 

S25 

S96 

S101 

S108 

S182 

LATsPPDsFLDDHHL 

DEItEAksGtAtPQR 

GtAtPQRsGsVsNYR 

AtPQRsGsVsNYRSC 

SDGEFLRtsCGsPNy 

EtPRARHtLDELNPQ 

AtPQRsGsVsNYRSC 

SDGEFLRtsCGsPNy 

VDPMKRAtIKDIREH 

HKtPRRDssGGTKDG 

sKLPLTRsHNNFVAI 

MVDSQKCsDVsELss 

SQKCsDVsELsssPP 

APAQARPtVFRWTGG 

NIIQVKKtDFEVFDA 

HKTPRRDssGGTKDG 

KTPRRDssGGTKDGD 

KEVYLSGsFNNWsKL 

SGsFNNWsKLPLTRs 

sKLPLTRsQNNFVAI 

DVSELSSsPPGPYHQ 

19376078 AMPK is a heterotrimeric complex 

composed of a catalytic α subunit and 

regulatory β and γ subunits, each of which 

is encoded by two or three distinct genes 

(α1, 2; β1, 2; γ1, 2, 3). The kinase is activated 

by an elevated AMP/ATP ratio due 

17728241 

17023420 

16340011 

9305909 

to cellular and environmental stress, such 

as heat shock, hypoxia, and ischemia. Accumulating 

evidence indicates that AMPK 

not only regulates the metabolism of fatty 

acids and glycogen, but also modulates 

protein synthesis and cell growth through 

EF2 and TSC2/mTOR pathways, as well 

as blood flow via eNOS/nNOS. AMPKA1 

phosphorylation is required for AMPK 

activation. 

19376078 AMPK is a heterotrimeric complex 

composed of a catalytic α subunit and 

regulatory β and γ subunits, each of which 

is encoded by two or three distinct genes 

(α1, 2; β1, 2; γ1, 2, 3). The kinase is activated 

by an elevated AMP/ATP ratio due 

9305909 

12764152 

9305909 

to cellular and environmental stress, such 

as heat shock, hypoxia, and ischemia. Accumulating 

evidence indicates that AMPK 

not only regulates the metabolism of fatty 

acids and glycogen, but also modulates 

protein synthesis and cell growth through 

EF2 and TSC2/mTOR pathways, as well as 

blood flow via eNOS/nNOS. The β1 subunit 

is post-translationally modified by multisite 

phosphorylation to regulate AMPK 

activation and localization. 

AS160 AMPKA2 mouse S711 S704 PSLHTSFsAPSFTAP 19923418 AS160 is a Rab GTPase-activating protein 

that regulates insulin-stimulated Glut4 

trafficking. Phosphorylation of AS160 

by AMPK is involved in the regulation of 

contraction-stimulated Glut4 translocation. 

CFTR AMPKA1 human S737 

S768 

S737 

S768 

EPLERRLsLVPDSEQ 

LQARRRQsVLNLMTH 

19095655 

19419994 

CFTR is a plasma membrane cyclic 

AMP activated chloride channel that is 

expressed in the epithelial cells of the lung 

and several other organs. CFTR channels 

are kept closed by AMPK mediated phosphorylation 

in non-stimulated epithelium. 

ChREBP AMPKA1 rat S568 S556 TLLRPPEsPDAVPEI 11724780 Carbohydrate-responsive element-binding 

protein (ChREBP) is a transcriptional 

repressor that regulates cellular energy homeostasis. 

AMPK phosphorylation inhibits 

the ability of CHREBP to bind DNA. 

CK1-E AMPKA1 human S389 S389 RGAPANVsssDLtGR 17525164 CK1-E (Casein Kinase I epsilon) is a 

member of a family of protein kinases 

implicated in multiple processes including 

DNA repair, cell morphology, and Wnt 

signaling. Multiple inhibitory autophosphorylation 

sites have been identified near 

the C-terminus of CK1-E, including an 

AMPK site that results in increased CK1-E 

activity. 

AMPK 

Substrates 

The table provides a list of substrates 

for AMPK, along with corresponding 

phosphorylation sites and references. 

This table was generated using 

PhosphoSitePlus ® , Cell Signaling 

Technology’s protein modification 

resource. See page 250 for more 

information on PhosphoSitePlus ® . 

© 2006–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Reuben Shaw, The Salk Institute for Biological Studies, La Jolla, CA, for reviewing this diagram. 





Substrate 

AMPKA 

Isoform 





CRY1 AMPKA1 mouse S71 S71 ANLRKLNsRLFVIRG 19833968 CRY1 is a member of the DNA photolyase 

class-1 family that acts as a regulator of 

the circadian clock. CRY1 is regulated in 

rhythmic fashion by AMPK phosphorylation-induced 

degradation. 

eEF2K AMPKA1 human S398 S398 DSLPSsPsSATPHSQ 14709557 Eukaryotic elongation factor 2 kinase 

(eEF2k) phosphorylates and inactivates 

eEF2, resulting in the inhibition of peptidechain 

elongation. AMPK phosphorylation of 

eEF2K increases its ability to phosphorylate 

eEF2. 

eNOS AMPKA1 cow S1179 S1177 TSRIRtQsFSLQERH 12107173 Endothelial nitric-oxide synthase (eNOS) 

AMPKA1 human S633 

S1177 

AMPKA1 rat T494 

S1176 

S633, 

S1177 

T495, 

S1177 

WRRKRKEssNTDSAG 

TsRIRtQsFsLQERQ 

TGITRKKtFKEVANA 

TSRIRTQsFsLQERQ 

12791703 

17276402 

20479254 

10025949 

catalyzes the production of nitric oxide 

(NO), a key regulator of blood pressure, 

vascular remodeling, and angiogenesis. 

eNOS is activated by AMPK phosphorylating 

Ser1177 in response to various stimuli. 

GABBR1 AMPKA1 rat S948 S918 ELRHQLQsRQQLRSR 17224405 The metabotropic GABA(B) receptor is 

coupled to G proteins that modulate slow 

inhibitory synaptic transmission. Functional 

GABA(B) receptors form heterodimers 

of GABA(B)R1 and GABA(B)R2 where 

GABA(B)R1 binds the GABA ligand and 

GABA(B)R2 is the primary G protein contact 

site. AMPK mediated phosphorylation 

of GABA receptors increases activity as 

part of a neuroprotective mechanism. 

GABBR2 AMPKA1 rat S783 S784 VTSVNQAsTSRLEGL 17224405 The metabotropic GABA(B) receptor is 

coupled to G proteins that modulate slow 

inhibitory synaptic transmission. Functional 

GABA(B) receptors form heterodimers 

of GABA(B)R1 and GABA(B)R2 where 

GABA(B)R1 binds the GABA ligand and 

GABA(B)R2 is the primary G protein contact 

site. AMPK mediated phosphorylation 

of GABA receptors increases activity as 

part of a neuroprotective mechanism. 

GBF1 AMPKA1 human T1337 T1337 GKIHRsAtDADVVNs 18063581 Golgi-specific brefeldin A resistance factor 

1 promotes guanine nucleotide exchange 

in the Golgi apparatus. GBF1 phosphorylation 

by AMPK occurs in response to low 

glucose, resulting in Golgi disassembly 

and lowered intracellular levels of ATP. 

GFAT AMPKA1 human S261 S261 CNLsRVDsttCLFPV 17941647 

19170765 

GFAT, glutamine:fructose-6-phosphate 

aminotransferase 1, is the rate-limiting 

enzyme of the hexosamine biosynthesis 

pathway generating the building blocks for 

protein and lipid glycosylation. GFAT activity 

is regulated by AMPK phosphorylation. 

GYS1 AMPKA1 rabbit S8 S8 MPLSRTLsVSsLPGL 2567185 Glycogen synthase 1 (GYS1) is a key 

enzyme in the regulation of glycogen 

synthesis in muscle. AMPK mediated phosphorylation 

leads to inactivation of GYS1. 

H2B AMPKA1 human S37 S37 RKRsRkEsysIyVyk 20647423 The nucleosome, made up of four core 

histone proteins (H2A, H2B, H3, and H4), 

is the primary building block of chromatin. 

In response to metabolic stress, AMPK is 

recruited to responsive genes and phosphorylates 

histone H2B at S37, activating 

transcription. 

HAS2 AMPKA2 human T110 T110 LQSVKRLtYPGIKVV 21228273 Hyaluronan synthase 2 (HAS2) regulates 

the synthesis of hyaluronan (HA), an extracellular 

matrix protein involved in cell 

motility, proliferation, tumorigenesis, and 

inflammation. HAS2 phosphorylation by 

AMPK results in a loss of HAS2 enzymatic 

activity and impaired HA regulated functions. 

HDAC5 

AMPKA1, 

AMPKA2 

human S259, 

S498 

S259 

S498 

FPLRKTAsEPNLKVR 

RPLSRtQssPLPQsP 

18184930 Histone deacetylase 5 (HDAC5) acts as a 

repressor of transcription by removing histone 

tail acetylations, promoting a closed 

chromatin configuration. AMPK mediated 

phosphorylation inhibits the repression 

activity of HDAC5. 

Substrate 

AMPKA 

Isoform 





HNF4α AMPKA1 human S313 S313 GkIkRLRsQVQVsLE 12740371 Hepatocyte nuclear factor 4α (HNF4α) is 

a transcription factor that belongs to the 

steroid hormone receptor superfamily and 

regulates lipid homeostasis in the liver. 

AMPK phosphorylation of HNF4α inhibits 

dimer formation and DNA binding, resulting 

in increased protein degradation. 

HSL AMPKA1 human S855 S855 EPMRRsVsEAALAQP 16188906, 

2537200 

IKKβ AMPKA2 human S177 

S181 

S177 

S181 

AKELDQGsLCtsFVG 

DQGsLCtsFVGTLQy 

HSL (hormone-sensitive lipase) catalyzes 

the hydrolysis of triacylglycerol, the ratelimiting 

step in lipolysis. AMPK phosphorylation 

of HSL reduces HSL phosphorylation 

by PKA and inhibits HSL activity. 

21673972 The NF-κB/Rel transcription factors are 

present in the cytosol in an inactive state, 

in a complex with the inhibitory IκB proteins. 

IκB kinase (IKK) complex containing 

the IKKβ catalytic subunit targets IκB for 

proteasomal degradation. Activation of IKK 

depends upon AMPK phosphorylation of 

the activation loop of IKKβ. 

IRS1 AMPKA1 mouse S789 S794 QHLRLSSsSGRLRYT 11598104 Insulin receptor substrate 1 (IRS1) is one 

of the major substrates of the insulin 

receptor kinase. Insulin signaling pathway 

activity is increased by AMPK phosphorylation 

of IRS1. 

KCNMA1 AMPKA1 mouse S722 S722 GRSERDCsCMSGRVR 21209098 Calcium-activated potassium channel 

subunit a-1 (KCNMA1) is a K+ channel 

activated by membrane depolarization, 

increased cytosolic Ca2+, and cytosolic 

Mg2+. KCNMA1 regulates several 

membrane polarization activities, as well 

as acting as an oxygen mediator under 

hypoxic conditions. KCNMA1 is inhibited 

by AMPK phosphorylation in cell types that 

do not monitor oxygen levels. 

Kir6.2 AMPKA1 rat S385 S385 AKPKFSIsPDSLS__ 19357830 ATP-sensitive inward rectifier potassium 

channel 11 (Kir6.2) is a G protein mediated 

receptor that allows K+ to flow into 

the cell. The Kir6.2 channel is closed by 

AMPK mediated phosphorylation to allow 

insulin secretion in pancreatic beta cells. 

KLC1 AMPKA1 human S521 S521 ENMEkRRsREsLNVD 20074060 Kinesin light chain 1 (KLC1), also known as 

KNS2, is a motor protein that associates 

with microtubule components of the 

cytoskeleton. The intracellular trafficking 

of organelles may be regulated by AMPK 

mediated phosphorylation of KLC1. 

KLC2 AMPKA1 human S545 

S582 

S545 

S582 

GSLRRsGsFGKLRDA 

PRMKRAssLNFLNKs 

21725060 Kinesin light chain 2 (KLC2) is a motor 

protein that associates with microtubule 

components of the cytoskeleton. The 

intracellular trafficking of organelles 

may be regulated by AMPK mediated 

phosphorylation of KLC2. 

KPNA2 AMPKA1 human S105 S105 QAARKLLsREkQPPI 15342649 Importin subunit a-2 (KPNA2) is an adaptor 

subunit of the Importin nuclear protein 

import receptor. KPNA2 phosphorylation 

by AMPK is required for Importin nuclear 

import mediation activity. 

Kv2.1 

AMPKA1 

AMPKA2 

rat 

S444 

S541 

S444, 

S541 

ERAKRNGsIVsMNMK 

SKMAKTQsQPILNTK 

22006306 Potassium voltage-gated channel subfamily 

B member 1 (Kv2.1) mediates voltage 

dependent flow of K+ across membranes. 

Kv2.1 mediated action potential frequency 

is modulated under stress conditions via 

phosphorylation by AMPK. 

mTOR AMPKA1 mouse T2446 T2446 NKRsRtRtDsysAGQ 14970221 The mammalian target of rapamycin 

(mTOR) is a Ser/Thr protein kinase that 

functions as an ATP and amino acid 

sensor to balance nutrient availability and 

cell growth. AMPK phosphorylates mTOR 

in response to nutrient deprivation and 

inhibits mTOR response to growth factor 

phosphorylation. 





AMPKA Published Data Human 


Substrate Isoform Organism Site Site Sequence (+/-7) PMID Effect of Phosphorylation 

NKCC2 AMPKA1 human S130 S130 GPKVNRPsLLEIHEQ 19176702 NKCC2 is an electroneutral cation chloridecoupled 

efflux cotransporter that regulates 

AMPKA1 mouse S126 S130 GPKVNRPsLLEIHEQ 17341212 

cell volume and maintains cellular homeostasis 

in response to osmotic and oxidative 

stress. NKCC2 chloride efflux activity 

is inhibited by AMPK phosphorylation, 

thereby increasing intracellular chloride 

concentration in the kidney. 

p27Kip1 AMPKA1 human T198 T198 PGLRRRQt______ 17237771 p27 Kip1 is a member of the Cip/Kip family 

of cyclin-dependent kinase inhibitors 

AMPKA1 mouse S83 S83 WQEVERGsLPEFyYR 17237771 

T170 T170 QNKRANRtEENVSDG 18701472 that enforces the G1 restriction point via 

T197 T198 KPGLRRQt______ 20146253 its inhibitory binding to CDK2/cyclin E and 

other CDK/cyclin complexes. p27Kip1 

stability is increased by AMPK mediated 

phosphorylation, resulting in increased 

survival under stress conditions. 

p300 AMPKA1 human S89 S89 SELLRSGsSPNLNMG 11518699 The transcriptional coactivator p300 associates 

with transcriptional regulators and 

signaling molecules, integrating multiple 

signal transduction pathways with the 

transcriptional machinery. AMPK mediated 

phosphorylation represses p300 activity 

by disrupting the association of p300 with 

nuclear receptors. 

p53 AMPKA1 human S20 

T18 


S20 

T18 

PLsQEtFsDLWKLLP 

EPPLsQEtFsDLWKL 

AMPKA2 mouse S15 S15 IsLELPLsQEtFsGL 15866171 

17339337 The p53 tumor suppressor protein plays 

a major role in cellular response to DNA 

damage and other genomic aberrations. 

Activation of p53 can lead to either cell 

cycle arrest and DNA repair or apoptosis. 

DNA damage induces phosphorylation of 

p53 and leads to a reduced interaction 

between p53 and its negative regulator, 

the oncoprotein MDM2. 

PFKFB2 AMPKA1 human S466 S466 PVRMRRNsFtPLSSS 12853467 Phosphofructokinase (PFK) catalyzes the 

phosphorylation of fructose-6-phosphate 

in glycolysis. PFKFB2 initiated glycolysis is 

activated by AMPK phosphorylation. 

PFKFB3 AMPKA1 human S461 S461 NPLMRRNsVtPLAsP 12065600, 

15896703 

PGC-1α AMPKA2 mouse T177 

S538 

T178 

S539 

NHTHRIRtNPAIVkT 

SLFDVSPsCSSFNSP 

Phosphofructokinase (PFK) catalyzes the 

phosphorylation of fructose-6-phosphate 

in glycolysis. PFKFB2 initiated glycolysis is 

activated by AMPK phosphorylation. 

17609368 PGC-1α interacts with a diverse array of 

transcription factors to regulate adaptive 

thermogenesis, energy metabolism, 

glucose uptake, gluconeogenesis, insulin 

secretion, and mitochondrial biogenesis. 

PGC-1α activity in skeletal muscle is 

induced by AMPK-mediated phosphorylation. 

PLD1 AMPKA2 human S505 S505 GSVKRVTsGPsLGSL 20231899 Phosphatidylcholine-specific phospholipase 

D (PLD) hydrolyzes phosphatidylcholine 

(PC) to produce choline and 

phosphatidic acid (PA). PA is the precursor 

of the second messenger, diacylglycerol 

(DAG). PLD1 is activated by AMPK 

phosphorylation, leading to an increase 

in glucose uptake in muscle under stress 

deprivation conditions. 

PPP1R3C AMPKA1 human S33 

S293 

PPP2R5C AMPKA1 human S298 

S336 

S33 

S293 

S298 

S336 

MRLCLAHsPPVKSFL 

LESTIFGsPRLASGL 

KYWPKTHsPKEVMFL 

RQLAKCVsSPHFQVA 

19171932 Protein phosphatase 1 is a serine/ 

threonine phosphatase holoenzyme 

composed of a catalytic subunit and an 

inhibitory regulatory subunit. PPP1R3C is a 

regulatory subunit that confers specificity 

for increasing glycogen synthesis. AMPK 

targets PPP1R3C for phosphorylation and 

proteasomal degradation, which inhibits 

glycogen synthesis. 

19366811 Protein phosphatase 2 is a tripartite 

serine/threonine phosphatase holoenzyme 

composed of a catalytic subunit, a 

structural subunit and a regulatory 

subunit. AMPK phosphorylates the regulatory 

subunit PPP2R5C, which results in 

dephosphorylation of the catalytic subunit 

and increased PP2A activity. 



Raf1 AMPKA1 human S259 

S621 

Human 


S259 

S621 

sQRQRststPNVHMV 

PKINRsAsEPsLHRA 



9091312 Raf-1 (c-Raf) is recruited by GTP-bound 

Ras to activate the MEK-MAP kinase 

pathway. Inhibitory 14-3-3 protein binding 

sites on c-Raf can be phosphorylated by 

AMPK. 

raptor AMPKA1 human S792 S792 DKMRRASsYSsLNsL 18439900 The regulatory associated protein of mTOR 

(Raptor) was identified as an mTOR binding 

partner that mediates mTOR signaling 

to downstream targets. AMPK phosphorylation 

of raptor is essential for inhibition 

of the raptor-containing mTOR complex 1 

(mTORC1) and induces cell cycle arrest 

when cells are stressed for energy. 

Rb AMPKA1 mouse S804 S811 IYIsPLKsPyKIsEG 19217427 The retinoblastoma tumor suppressor 

protein, Rb, regulates cell proliferation by 

controlling progression through the restriction 

point within the G1-phase of the cell 

cycle. AMPK regulation of brain development 

is achieved by modulating control of 

the cell cycle via phosphorylation of Rb. 

smMLCK AMPKA1 chicken S1749 S1760 RAIGRLSsMAMISGM 18426792 Smooth muscle myosin light chain kinase 

(smMLCK) is activated by high Ca 2+ induced 

calcium/calmodulin. Smooth muscle 

contraction is activated smMLCK mediated 

phosphorylation of myosin light chains. 

Smooth muscle contraction is attenuated 

by AMPK phosphorylation and inactivation 

of smMLCK. 

TBC1D1 AMPKA1 human S237 

T596 

S237 

T596 

RPMRKSFsQPGLRsL 

AFRRRANtLsHFPIE 

17995453 TBC1D1 is a Rab GTPase activating protein 

involved in vesicle trafficking in response 

to insulin. AMPK acts in association with 

insulin and growth factor signaling to activate 

TBC1D1 mediated vesicle regulation. 

TIF-IA AMPKA1 human S635 S635 DTHFRsPsSSVGsPP 19815529 RNA polymerase I-specific transcription 

initiation factor RRN3 (TIF-IA) is required 

for RNA polymerase I initiation. Transcription 

of rRNA is inhibited during times of 

stress by AMPK phosphorylation inhibition 

of TIF-IA. 

TORC2 AMPKA1 mouse S171 S171 SALNRtssDsALHTs 16148943 Torc2 (transducer of regulated CREB activity 

2) functions as a CREB co-activator and 

is implicated in mediating the effects of 

hormone and glucose sensing pathways. 

Torc2 is phosphorylated by AMPK at 

Ser171 and becomes sequestered in the 

cytoplasm, inhibiting hepatic gluconeogenesis. 

TSC2 AMPKA1 rat S1389 

T1271 

S1387, 

T1271 

QPLsKSSsSPELQTL 

PTLPRSNtVASFSSL 

16959574 

14651849 

Tuberin is a product of the TSC2 tumor 

suppressor gene and an important regulator 

of cell proliferation and tumor development. 

AMPK phosphorylates tuberin during 

periods of energy starvation, enhancing 

tuberin activity and resulting in increased 

translation. 

ULK1 AMPKA1 human S638 S638 FDFPKtPssQNLLAL 21383122 ULK1 is a serine/threonine kinase involved 

AMPKA1 mouse S467 S467, sAIRRsGsttPLGFG 21205641 in axon growth, endocytosis of critical 

S555 S556 GLGCRLHsAPNLSDF 

growth factors such as NGF, and can act 

as a convergence point for multiple signals 

that control autophagy. AMPK, activated 

during low nutrient conditions, directly 

phosphorylates ULK1 at multiple sites to 

promote autophagy. 

VASP AMPKA1 human T278 T278 LARRRKAtQVGEktP 17082196 VASP belongs to the Ena/VASP family of 

adaptor proteins linking the cytoskeletal 

system to the signal transduction pathways 

and that it functions in cytoskeletal 

organization, fibroblast migration, platelet 

activation, and axon guidance. AMPK 

phosphorylation of VASP leads to specific 

cytoskeletal rearrangements. 

ZNF692 AMPKA1 human S470 S470 VAAHRSKsHPALLLA 17097062 ZNF692, also known as AREBP, is a zinc 

finger transcription factor involved in the 

expression of gluconeogenesis genes. 

ZNF692 DNA binding ability is abrogated 

by AMPK mediated phosphorylation during 

times of metabolic stress. 


06 


ESC Signaling 

ESCs can be identified using markers specific 

to the pluripotency transcription factors, the 

cell surface glycoproteins SSEA3/4, and the 

TRA-1-60 and TRA-1-81 antigens. 

Development and Differentiation 

Embryogenesis is a complex and highly ordered program of proliferation and directed differentiation, 

during which a fertilized egg undergoes transformation from an undifferentiated single-cell entity 

(zygote) into a multicellular organism composed of numerous, functionally-distinct cell types. This 

transformation is under the control of an intricate array of epigenetic, genetic, and biochemical signaling 

cascades that collectively influence the fate and behavior of individual cells in a developmentally 

appropriate context. 

Embryonic Stem Cells 

ESCs 

Embryonic stem cells (ESCs) are derived from the inner cell mass of a cleavage-stage embryo, or blastocyst. 

ESCs exhibit a suite of distinct properties that make them an exceptionally powerful research 

tool in developmental biology and a source of significant therapeutic potential in regenerative medicine. 

These features include (1) pluripotency, defined as the ability to differentiate into any cell lineage of the 

body; and (2) the capacity for self-renewal, which allows them to be propagated indefinitely in culture. 

Distinct cell signaling pathways are responsible for endowing and maintaining these properties in 

ESCs. In humans, foremost among these are the BMP/TGF-β signaling pathway, which signals through 

SMAD proteins, and the FGF signaling pathway, which activates MAPK and Akt pathways. The Wnt signaling 

pathway has also been implicated in the maintenance of pluripotency, through a noncanonical 

Wnt signaling mechanism that serves to maintain a balance between the transcriptional activator TCF1 

and the repressor TCF3. Likewise, the Hippo signaling pathway also plays an important role in ESC 

biology. Down-regulation of the transcriptional co-activator YAP, a hallmark of Hippo pathway activation, 

results in a loss of ESC pluripotency, whereas over-expression of YAP prevents ESC differentiation, 

even in culture conditions that otherwise promote differentiation. The role of NOTCH signaling in ESC 

biology is somewhat less clear. Human (but not mouse) ESCs have an active Notch signaling pathway, 

and Notch inactivation results in reduced human ESC proliferation in culture, suggesting that Notch signaling 

may be required for ESC proliferation. This mechanism, however, does not appear to be widely 

conserved among mammals. 

membrane 

cytoplasm 

FGF2 

IGF 

LRP 

Wnt 

TGF-β/ 

Activin/Nodal 

BMP4 

Cell-Cell 

Contact 

iPSCs 

Induced pluripotent stem cells (iPSCs) are pluripotent, ESC-like cells that can be experimentally derived 

from differentiated cells by forced expression of a defined set of reprogramming factors, the best known 

of which are Oct-4, Sox2, KLF4, and c-Myc (commonly known as the Yamanaka factors, after Shinya 

Yamanaka, the scientist who first generated iPSCs in 2006 [Takahashi, K. and Yamanaka, S. (2006) 

Cell 126, 663–676.]. In vitro reprogrammed iPSCs exhibit a gene expression signature similar to that 

of ESCs and, like their embryonic counterparts, are the subject of intense investigation due to their 

enormous potential for use in regenerative medicine, drug screening, and basic developmental biology 

research. Alternatively, iPSCs may be generated by autologous somatic cell nuclear transfer (SCNT). 

There is considerable interest in this approach, as SCNT-derived pluripotent cells exhibit an epigenetic 

signature that more closely resembles ESCs than do iPSCs generated by in vitro reprogramming. 

A B C 

D E F 

Transcription Factors and Development 

Transcription factors play an instrumental role in both pluripotency and cell fate specification (differentiation) 

by regulating the global expression patterns of developmentally important genes. In ESCs 

and iPSCs, pluripotency is induced and maintained by the expression of three key transcription factors: 

Oct-4, Sox2, and Nanog. These transcription factors promote a pluripotency gene expression signature, 

regulate their own expression through an auto-regulatory loop, and serve as important biochemical 

or molecular markers of pluripotency. Research studies are revealing a complex network of additional 

transcription factors that function to maintain pluripotency, through transcriptional regulation of the core 

pluripotency factor network. 

chapter 06: Development and differentiation 

Expression of 

pluripotency markers 

in human iPSCs 

StemLight Pluripotency Antibody 

Kit #9656: Projected confocal z-stack 

of human iPS cells using TRA-1-60(S) 

(TRA-1-60(S)) Mouse mAb (A), TRA-1- 

81 (TRA-1-81) Mouse mAb (B), SSEA4 

(MC813) Mouse mAb (C), Oct-4A 

(C30A3) Rabbit mAb (D), Sox2 (D6D9) 

XP ® Rabbit mAb (E) and Nanog (D73G4) 

XP ® Rabbit mAb (F). Actin filaments 




nucleus 

PI3K 

Akt 

Erk1/2 

β-catenin Smad2/3 Smad1/5/9 


Oct-4 

Sox2 

Oct-4 Sox2 FoxD3 

Oct-4 Sox2 Nanog 

Oct-4, 

Sox2, 

FoxD3, 

FGF4 

Nanog 

gene 

expression 

YAP 

Pluripotency 

and Self Renewal 

hESC Markers: 

Oct-4 

Nanog 

Sox2 

SSEA 3/4 

TRA-1-60 

TRA-1-81 

Oct-4, Sox2, and Nanog pluripotency factors regulate 

their expression through an auto-regulatory network. 

SimpleChIP ® Stem Cell Master Regulator Assay Kit #8980: 

Chromatin IPs were performed with cross-linked chromatin 

from 4 x 10 6 NCCIT cells and 10 μl of Nanog, Oct-4, and Sox2 

antibodies or 2 μl of Normal Rabbit IgG, using SimpleChIP ® 

Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The 

enriched DNA was quantified by real-time PCR using human 

Nanog promoter primers, SimpleChIP ® Human Oct-4 Promoter 

Primers #4641, SimpleChIP ® Human Sox2 Promoter Primers 

#4649, and SimpleChIP ® Human α Satellite Repeat Primers 

#4486. The amount of immunoprecipitated DNA in each sample 

is represented as a percent of the total input chromatin. 


3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0 

Nanog Oct-4 Sox2 α Satellite 

Generation of Induced Pluripotent Stem Cells 

Download a free copy of ‘Generation of Induced Pluripotent Stem Cells’, a white paper written by Alison Becker 

from CST in collaboration with Ryan M. Walsh and Konrad Hochedlinger of the Harvard Stem Cell Institute. 

www.cellsignal.com/cstliterature 

Nanog (D73G4) XP ® Rabbit mAb 

(ChIP Formulated) #5232 

Oct-4A (C30A3C1) Rabbit mAb 


Sox2 (D6D9) XP ® Rabbit mAb 


Normal Rabbit IgG #2729 

www.cellsignal.com/cstdevelopment 153



GATA-6, a zinc finger 

protein that regulates 

endoderm development, 

is expressed in colon 

carcinoma (KM12) but 

not ovarian carcinoma 

(SK-OV-3) cells. 

Events 

Sox2 (Alexa Fluor ® 647 Conjugate) 

Pluripotency marker Sox2 is expressed 

in embryonic carcinoma NTERA-2 cells. 

Sox2 (D6D9) XP ® Rabbit mAb (Alexa Fluor ® 647 Conjugate) #5067: 

Flow cytometric analysis of HeLa cells (blue) and NTERA-2 cells (green) 

using #5067. 

While much of modern stem cell research has focused on the isolation or generation of pluripotent 

stem cells, more recent efforts have been directed towards understanding and exploiting the process 

of transdifferentiation, or direct reprogramming. Transdifferentiation describes a process whereby a 

terminally differentiated cell is converted into a functionally distinct cell type, without first proceeding 

through a pluripotent phase of development. Transdifferentiation is considered a promising avenue 

of exploration in regenerative medicine, as transdifferentiated cells could provide for direct in situ 

replacement of damaged cells without the need for in vitro differentiation, as required by iPSCs. Transdifferentiation 

can be experimentally induced by the forced expression of a combination of transcription 

factors, unique for each cell type. For example, mouse cardiac fibroblasts can be transdifferentiated 

into cardiomyocytes by forced expression of GATA-4, MEF2C, and Tbx5 (Qian, L. et al. (2012) Nature 

485, 593–598.). 

Miwi is expressed in 

mouse testes and is a 

marker for germ cells. 

Several additional transcription factor families regulate differentiation along lineage-specific pathways. 

These include members of the zinc finger, homeobox, forkhead box (Fox), helix-loop-helix (bHLH), T 

box, Paired box (Pax), and Sox protein families. Members within each group are classified by the presence 

of unique DNA binding domains, which bind to specific promoter regions to regulate expression 

of genes necessary for each stage of development. One example are the GATA proteins, an extremely 

large, highly conserved family of zinc finger proteins, the members of which play diverse and critically 

important roles throughout development. Similarly, homeobox transcription factors, the product of Hox 

genes, contain a helix-turn-helix homeodomain and are critical for regulating the transcription of genes 

that specify development along the anterior-posterior body axis. 

Germ cell marker DDX4 is expressed in mouse testes but not mouse brain. 

DDX4 (D10C5) Rabbit mAb #8761: 

Confocal IF analysis of mouse testes 

(left) and mouse brain (right) using 





Miwi (D92B7) XP ® Rabbit mAb #6915: 

Confocal IF analysis of mouse testes 





GATA-6 (D61E4) XP ® Rabbit mAb 

#5851: Confocal IF analysis of KM12 (top) 

and SK-OV-3 (bottom) cells using #5851 


DY-554 phalloidin (red). 

Stem Cell Differentiation and Transdifferentiation 

Pluripotent stem/progenitor cells, including ESCs and iPSCs, can be induced to develop into lineagespecific 

progenitor cells, representing each of the three primary germ layers established during 

gastrulation: ectoderm, mesoderm, and endoderm. Further differentiation then proceeds progressively 

along the respective lineage-specific pathways, culminating in terminal differentiation and yielding a 

cell with a lineage-specific functional phenotype. Cells that have differentiated into a specific lineage 

may be identified using lineage markers—antigens with a spatially or temporally restricted pattern of 

expression that can be used to identify cells within specific lineage pathways. 

Neurofilament-L (C28E10) Rabbit 

mAb #2837 and β3-Tubulin (TU-20) 

Mouse mAb #4466: Confocal IF 

analysis of neuroepithelial clusters 

differentiated from human iPS cells, 

showing multiple neurite extensions, 

using #2837 (red) and #4466 (green). 



Neurofilament-L 

and β3-Tubulin 

are markers for 

neuronal cells and 

antibodies for these 

proteins label cells 

in neuroepithelial 

clusters. 

Induced Pluripotency (iPS) 

Embryonic Stem Cell 

Primordial Germ Cell 

Ectoderm 

Mesoderm 

Endoderm 

Development and Differentiation Signaling 

Development along each lineage is regulated by several signaling pathways that control cell division, 

growth, and differentiation, including BMP/TGF-β, Notch, Wnt/β-catenin, Hedgehog, and Hippo pathways. 

Each of these pathways is regulated by a complex array of genetic, epigenetic, and exogenous 

signaling factors that serve to guide cell fate and behavior during development and differentiation. 

Additional details of signaling nodes within each of these pathways can be found in the pathway 

diagrams to follow. 

β-catenin is 

abundantly expressed 

in the mammalian gut, 

where it regulates 

epithelial cell adhesion. 

Neural Stem Cell 

Mesenchymal 

Stem Cell 

Neural Crest Glial 

Neuron 

Adipocyte, Hematopoietic Hepatocyle Pancreatic Cell 

Progenitor Progenitor Myocyte, Osteocyte Stem Cell 

Astrocyte Oligodendrocyte Neuron 

Hemangioblast 


Endodermal 

Progenitor 

Wnt/β-catenin Signaling 

The widely conserved Wnt/β-Catenin pathway regulates stem cell pluripotency and cell fate decisions 

during development. Wnt signaling is triggered by binding of the Wnt ligand to Frizzled receptors in 

complex with the co-receptor LRP5/6, initiating a signaling cascade that results in stabilization and 

nuclear translocation of the transcriptional co-regulator, β-catenin. Nuclear β-catenin functions as a 

transcriptional co-activator, promoting the transcription of genes that regulate proliferation and differentiation. 

β-catenin also plays a highly important role in the planar-cell-polarity pathway, regulating 

cell-cell contact via adherens junctions. 

β-Catenin (D10A8) XP ® Rabbit mAb 


colon using #8480 (green). Actin filaments 








YAP localizes to 

the nucleus in low 

confluence cells, but 

is translocated to the 

cytoplasm during 

high confluence 

(contact inhibition). 

Hippo Signaling 

Hippo signaling is an evolutionarily conserved pathway that plays a critical role in development by 

regulating cell proliferation, apoptosis, and stem cell self-renewal. Activation of the Hippo pathway 

(e.g., by cell-cell contact) suppresses cell proliferation by initiating a signaling cascade that results in 

phosphorylation, cytoplasmic retention, and proteasomal degradation of the transcriptional co-activators 

YAP and TAZ, thereby preventing the transcription of genes that promote proliferation. 

A B C 

Notch Signaling 

The Notch pathway is a contact-dependent signaling cascade mediated by the Notch receptor on the 

receiving cell and the Delta-like and Jagged ligands on the signal-sending cell. Receptor-ligand binding 

results in a series of cleavages to the Notch receptor, culminating in release of the Notch intracellular 

domain (NICD) by γ-secretase. The NICD translocates to the nucleus where it interacts with RBPSUH 

and Mastermind-like (MAML) proteins to regulate transcription of target genes that mediate cell fate 

decisions in neuronal, cardiac, immune, and endocrine development. 

Hedgehog Signaling 

The evolutionarily conserved Hedgehog (Hh) pathway plays a critical role in a time and positiondependent 

fashion during development. Hedgehog ligands (Sonic, Desert, and Indian Hedgehog) act as 

classic morphogens, whose concentration gradient promotes specific developmental outcomes at distinct 

concentration thresholds. Hedgehog signaling occurs when a hedgehog family ligand binds to the 

receptor Patched (PTCH), releasing Smoothened (SMO) from suppression and allowing it to translocate to 

primary cilium. Once there, SMO signals through G proteins, ultimately leading to nuclear translocation 

of GLI zinc finger transcription factors. Hedgehog signaling is instrumental early in embryogenesis, 

orchestrating neural plate patterning and ventral polarity of the neural tube, and anterior-posterior axis 

patterning (e.g. during limb formation). Defects in Hh signaling are the most common cause of midline 

birth defects, resulting in disorders such as holoprosencephaly, which is characterized by incomplete 

separation of the forebrain hemispheres and their derivatives. The Hedgehog pathway has also been 

shown to play a role in maintenance of adult stem cells, particularly neural progenitors and hematopoietic 

stem cells. 

YAP (D8H1X) XP ® Rabbit mAb #14074: Confocal IF analysis of low confluence MCF 10A cells (A), high confluence MCF 10A (B), and 

YAP-negative RL-7 cells (C) using #14074 (green). Blue pseudocolor in lower images = DRAQ5 ® #4084 (fluorescent DNA dye). Increased 

nuclear localization of YAP protein is seen in low confluence (proliferating) cells. 

TGF-β/BMP Signaling 

Transforming growth factor-β (TGF-β) superfamily signaling plays a critical role in the regulation of cell 

proliferation, differentiation, developmental patterning and morphogenesis, and disease pathogenesis. 

In the canonical TGF-β signaling pathway, ligand binding to one of the three types of TGF-β receptors 

results in receptor activation and phosphorylation of the Smad2/3 or Smad1/5/9 effector proteins. 

These phosphorylated Smads dimerize with the co-activating Smad4 and translocate to the nucleus, 

where they stimulate transcription of target genes. TGF-β receptors can also signal through noncanonical 

(Smad-independent) pathways that promote cytoskeletal rearrangement and adhesion through 

activation of RhoA, Rac/cdc42, and PI3K pathways. 

BMP2 treatment results in phosphorylation 

of Smad1/5 at Ser463/465. 

Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad9 (Ser465/467) 

(D5B10) Rabbit mAb #13820: WB analysis of extracts from Hep G2 or MEF cells, 

untreated (-) or treated with Human BMP2 #4697 (50 ng/ml, 30 min; +), using 

#13820 (upper) and Smad1 (D59D7) XP ® Rabbit mAb #6944 (lower). 

Lanes 

1. Hep G2 

2. MEF 

kDa 

140 

100 

80 

60 

50 

40 

30 

20 

140 

100 

80 

60 

50 

40 

30 

1 2 

Phospho-Smad1 

(Ser463/465)/ 

Smad5 (Ser463/465)/ 

Smad9 (Ser465/467) 

Smad1 

Development and Cancer 

Ectopic activation or dysregulation of embryonic signaling pathways is recognized as an important 

contributor to tumorigenesis in many contexts. For example, constitutive activation of the Hedgehog 

pathway by mutations in PTCH or SMO are strongly linked to basal cell carcinoma and medulloblastoma. 

Likewise, mutations in components of the Wnt signaling pathway, which can lead to increased stability 

and transcriptional activity of β-catenin, are directly associated with colorectal, gastric, hepatocellular, 

ovarian, breast, and prostrate cancers. In Hippo signaling, the central pathway mediators, YAP and 

TAZ, function as oncogenes, and amplification of YAP has been associated with a variety of tumors 

including hepatocellular, lung, colorectal, breast, and liver carcinomas. The TGF-β signaling pathway 

is recognized as a potentially important driver of cancer metastasis, through its ability to regulate the 

process of epithelial-mesenchymal transition (EMT). 

Cancer stem cells (CSC) are a rare sub-population of tumorigenic cells found within tumors that (like 

normal stem cells) are multipotent and self-renewing. These cells posses tumor-initiating potential and 

can differentiate into all the other cell types that compose the bulk of the tumor. Cell surface markers 

can distinguish CSCs from differentiated cancer cells in many tumor types and be used to purify subpopulations 

of CSCs. These include CD133, CD44, CD24, ALDH1, and EpCAM, among others. 


Addis, R.C. and Epstein, J.A. (2013) Nat. Med. 19, 829−836. • Ader, M. and Tanaka, E.M. (2014) Curr. Opin. Cell Biol. 31C, 

23−28. • Gieseck, R.L. 3rd, Colquhoun, J., and Hannan, N.R. (2014) Biochim. Biophys. Acta. Jun 2 [Epub ahead of print] • 

Ingham, P.W. and McMahon, A.P. (2001) Genes and Dev. 15, 3059−3087. • Isobe, K.I., Cheng, Z., Nishio, N. et al. (2014) Nat. 

Biotechnol. 31, 411−421. • Itoh, F., Watabe, T., and Miyazono, K. (2014) Semin. Cell Dev. Biol. 32C, 98−106. • Jamieson, 

C., Sharma, M., and Henderson, B.R. (2014) Semin. Cancer Biol. 27C, 20−29. • Jiang, J. and Hui, C-C. (2008) Developmental 

Cell 15, 801−812. • Jopling, C., Boue, S., and Izpisua Belmonte, J.C. (2011) Nat. Rev. Mol. Cell Biol. 12, 79−89. • Kreso, 

A. and Dickemail, J.E. (2014) Cell Stem Cell 14, 275–291. • Lien, W.H. and Fuchs, E. (2014) Genes Dev. 28, 1517−1532. • 

Ntziachristos, P., Lim, J.S., Sage, J., et al. (2014) Cancer Cell 25, 318−334. • Robbins, D.J., Fei, D.L., and Riobo, N.A. (2012) 

Sci. Signal. 5, re6. • Sánchez, A.A. and Yamanaka, S. (2014) Cell 157, 110−119. • Sarkar, A. and Hochedlinger, K. (2013) 

Cell Stem Cell 12, 15−30. • Torres-Padilla, M.E. and Chambers, I. (2014) Development 141, 2173−2181. • Varelas, X. 

(2014) Development 141, 1614−1626. 

Elevated levels of 

β-catenin are found in 

colon carcinoma. 

β-Catenin (D10A8) XP ® Rabbit mAb 


human colon carcinoma using #8480. 

Amplification of YAP is 

associated with breast 

adenocarcinoma. 

20 

– + 

– + 

Human BMP2 

Cancer Research 

Please visit our website to learn more about the scientific tools and educational resources we have online for cancer 

signaling and proteomic analysis, including discussion of key disease drivers. www.cellsignal.com/cancerguide 

YAP (D8H1X) XP ® Rabbit mAb #14074: 

IHC analysis of paraffin-embedded human 

breast adenocarcinoma using #14074. 


www.cellsignal.com/cstdevelopment 

157




nodes within development and differentiation 

signaling pathways and are 

commonly studied in development and 

stem cell research. Primary antibodies, 









Commonly Studied Development and Differentiation Targets 

Target M P C 

ACVR1 

AFP 

• 

• • • 

AGR2 • 

ALPP • 

Angiopoietin-2 • 

APC 

• 

Axin1 • 

Axin2 • • 

Phospho-BCL9L 

(Ser915) 

• 

Bcl-11B • 

BMP4 • 

BMP7 

• 

BMPR2 

• 

Brachyury • 

CACYBP • • 

• 

β-Catenin (Aminoterminal 

Antigen) 

β-Catenin (Carboxyterminal 

Antigen) 

β-Catenin 

Phospho-β-Catenin 

(Ser33/37) 


(Ser33/37/Thr41) 

Non-phospho (Active) 

β-Catenin (Ser33/37/ 

Thr41) 


(Thr41/Ser45) 


(Ser45) 


(Ser552) 


(Ser675) 

• 

• • • 

• 

• 

• • • 

• • 

• 

• 

• • 

Acetyl-β-Catenin (Lys49) • • 

CDCP1 • 

• 

Phospho-CDCP1 

(Tyr707) 

Phospho-CDCP1 

(Tyr734) 

Phospho-CDCP1 

(Tyr743) 

Phospho-CDCP1 

(Tyr806) 

CDX2 

CEACAM1 

CK1 

Cripto 

CtBP1 

CtBP2 

DAX1 

DAZL 

DDX4 

DKK1 

DKK2 

DLK1 

DLL1 

DLL3 

• 

• 

• 

• • 

• 

• 

• • 

• 

• 

• 

• • 

• • 

• 

• 

• 

• 

• 

Target M P C 

DLL4 

• 

Deltex-2 

• 

Dvl2 

• • 

Dvl3 

• 

Endoglin • • 

EOMES 

• 

E-Ras • 

Acidic FGF • 

Basic FGF • 

FIH 

• 

FLI1 

• 

Flightless-I • 

FoxC1 • • 

FoxD3 • 

FoxK1 

• 

FoxK2 

• 

FoxP1 • • 

FoxP2 • 

Fragilis 

• 

Frizzled5 • • 

Frizzled6 • 

GATA-1 • • • 

GATA-3 • • 

GATA-6 • • 

GCNF/NR6A1 • 

GFI1b • 

GLI1 • • • 

GLI2 

• 

Gremlin 

• 

Heregulin • 

HES1 • 

HIF-1β/ARNT • • 

HIF-1α • 

Hydroxy-HIF-1α (Pro564) • 

HIF-2α • 

Id2 

• 

ID3 • • 

Jagged1 • 

Jagged2 • 

KIBRA 

• 

KIF3A • 

KIF3B 

• 

KLF4 • • 

LEF1 • • 

Lefty1 • 

LIMD1 

• 

LIN28A • • • 

LIN28B • • 

Lmx1B • 

LRP5 • 

LRP6 • 

Phospho-LRP6 

(Ser1490) • 

MAML1 • • 

MAML2 • • 

MESD2 • 

Mic-1 • • 

Target M P C 

MIS-R2 

• 

MITF • 

MOB1 • • 

Phospho-MOB1 (Thr12) • 

Phospho-MOB1 (Thr35) • 

Msx1 

• 

c-Myb • 

MyoD1 • • 

NAC1 

• 

Naked1 • • 

Naked2 • 

Nanog • • • 

NDRG1 • • 

Phospho-NDRG1 

(Ser330) • • 

Phospho-NDRG1 

(Thr346) • • • 

NDRG2 

• 

NDRG3 

• 

NDRG4 • 

NKX2.2 

• 

NKX2.5 • 

Notch1 • 

Cleaved Notch1 

(Val1754) • • 

Notch2 • 

Notch3 • • 

Notch4 • 

Nucleostemin • 

Numb • • 

Phospho-Numb (Ser276) • • 

Oct-1 • • 

Oct-4 • • • 

OTX2 • 

PAX2 

• 

PAX3 

• 

PAX5 • • 

PAX8 

• 

PAX9 • 

PDAP1 

• 

PHD-2/Egln1 • • 

PTCH1 • 

PTCH2 

• 

PTPN14 

• 

PTPN18 • 

RAIG1 • • 

RBPSUH • • 

RUNX2 • 

RUNX3/AML2 • 

Sall4 • • 

Sara 

• 

SAV1 • • 

Scribble 

• 

Phospho-Scribble • 

SCF 

• 

SFRP1 • • 

Shh 

• • 

Target M P C 

Shh/Ihh 

• 

SIX1 

• 

Slug 

• 

Smad1 • • 

Phospho-Smad1 

(Ser206) • • 

Phospho-Smad1 

(Ser463/465)/Smad5 

(Ser463/465)/Smad9 • • • 

(Ser426/428) 

Phospho-Smad1/5 

(Ser463/465) • • 

Smad2 • 

• 

Phospho-Smad2 

(Ser245/250/255) 

Phospho-Smad2 

(Ser465/467) 

Smad2/3 

Phospho-Smad2 

(Ser465/467)/Smad3 

(Ser423/425) 

Smad3 

Phospho-Smad3 

(Ser423/425) 

Smad4 

Smad5 

Smurf1 

Smurf2 

• • • 

• • • 

• • 

• • 

• 

• 

• • 

• 

• 


Palla, A.R. et al. (2014) Reprogramming activity of 

NANOGP8, a NANOG family member widely expressed in 

cancer. Oncogene 33, 2513–2519. 

Zhou, W. et al. (2014) Snail contributes to the maintenance 

of stem cell-like phenotype cells in human pancreatic 

cancer. PLoS One 9, e87409. 

Ono, T. et al. (2014) A single-cell and feeder-free culture system 

for monkey embryonic stem cells. PLoS One 9, e88346. 

Yan, X. et al. (2014) 5-azacytidine improves the osteogenic 

differentiation potential of aged human adipose-derived 

mesenchymal stem cells by DNA demethylation. PLoS One 

9, e90846. 

Han, H. et al. (2013) MBNL proteins repress ES-cellspecific 

alternative splicing and reprogramming. Nature 498, 

241–245. 

Hamalainen, R.H. et al. (2013) Tissue- and cell-type-specific 

manifestations of heteroplasmic mtDNA 3243A>G mutation 

in human induced pluripotent stem cell-derived disease 

model. Proc. Natl. Acad. Sci. USA 110, 3622–3630. 

Zhang, J. et al. (2013) NANOG modulates stemness in human 

colorectal cancer. Oncogene 32, 4397–4405. 

Jinesh, G.G. et al. (2013) Blebbishields, the emergency 

program for cancer stem cells: sphere formation and tumorigenesis 

after apoptosis. Cell Death Differ. 20, 382–395. 

Kregel, S. et al. (2013) Sox2 is an androgen receptorrepressed 

gene that promotes castration-resistant prostate 

cancer. PLoS One 8, e53701. 

Demir, K. et al. (2013) RAB8B is required for activity and 

caveolar endocytosis of LRP6. Cell Rep. 4, 1224–1234. 

Target M P C 

Snail • 

SnoN 

• 

Sox1 

• 

Sox2 • • • 

SPARC • • 

SSEA1 • 

SSEA4 • 

SUFU • • 

TACE • • 

TAZ 

• 

TAZ/YAP • 

TCF1 • • 

TCF3 • 

TCF4 • 

TCF8 • 

TCF12/HEB • 

TEAD1 • • 

Pan-TEAD • 

TEAD2 

• 

TEAD3 

• 

TFF1/pS2 • 

Pro-TGF-α • 

TGF-β • • 

TGF-β Receptor I • 

TGF-β Receptor II • 

Target M P C 

TGF-β Receptor III • • 

Thap11/Ronin • 

TLE1/2/3/4 • 

TRA-1-60 • 

TRA-1-81 • 

TRA-2-54 (Alkaline 

phosphatase) • 

TRIB2 • 

TRIM33 • 

Thyroid Transcription 

Factor 1 (TTF-1) • 

Phospho-TTF1 (Ser327) • 

UTF1 

• 

VEGF-B 

• 

VEGF-C 

• 

WBP2 

• 

WIF1 • • 

Wnt3a • • 

Wnt5a 

• 

Wnt5a/b • 

WTX • 

YAP 

• • 

Phospho-YAP (Ser127) • • 

Phospho-YAP (Ser397) • 

ZFX 

• 

Garg, N. et al. (2013) microRNA-17-92 cluster is a 

direct Nanog target and controls neural stem cell through 

Trp53inp1. EMBO J. 32, 2819–2832. 

Bhatia, S. et al. (2013) Demarcation of stable subpopulations 

within the pluripotent hESC compartment. PLoS One 

8, e57276 

Vaira, V. et al. (2013) Regulation of lung cancer metastasis 

by Klf4-Numb-like signaling. Cancer Res. 73, 2695–2705. 

Tanaka, A. et al. (2013) Efficient and reproducible myogenic 

differentiation from human iPS cells: prospects for modeling 

Miyoshi Myopathy in vitro. PLoS One 8, e61540. 

Wang, T. et al. (2013) Subtelomeric hotspots of aberrant 

5-hydroxymethylcytosine-mediated epigenetic modifications 

during reprogramming to pluripotency. Nat. Cell Biol. 15, 

700–711. 

Romorini, L. et al. (2013) Effect of antibiotics against Mycoplasma 

sp. on human embryonic stem cells undifferentiated 

status, pluripotency, cell viability and growth. PLoS One 8, 

e70267. 

Abad, M. et al. (2013) Reprogramming in vivo produces 

teratomas and iPS cells with totipotency features. Nature 

502, 340–345. 

Hasmim, M. et al. (2013) Cutting edge: Hypoxia-induced 

Nanog favors the intratumoral infiltration of regulatory T 

cells and macrophages via direct regulation of TGF-beta1. J. 

Immunol. 191, 5802–5806. 

Abhold, E.L. et al. (2012) EGFR kinase promotes acquisition 

of stem cell-like properties: a potential therapeutic target in 

head and neck squamous cell carcinoma stem cells. PLoS 

One 7, e32459. 

24 


CST antibodies for Nanog have been 








Wnt/b-Catenin Signaling 

Notch Signaling 

OFF-State 

Cadherin 

β-catenin 

α-catenin 

p120 

DKKs 

LRP5/6 

SFRP 

Frizzled 

ON-State 

Porc WIF 

WLS 

SFRP 

Wnt 

LRP5/6 

CK1 GSK-3 

Axin 

Frizzled 

Dvl 

Naked 

R-spondins 

LGR5/6 

RNF43 

ZNRF3 

Signal 

Sending 

Cell 

Inactive 

Ligand 

Delta 

Jagged 

Active 

Ligand 

Neur 

or Mib 

ub 

ub 

Delta 

Jagged 

Endosome 

ub 

Ligand 

Repositioning 

Epsin 

ub 

Ligand-receptor 


Skp 

Proteasomal 

Degradation 

β-TrCP 

ub β-catenin 

Tankyrase 

PP2A 

Axin 

APC GSK-3 

WTX 

β-catenin 

CK1 

CYLD 

β-catenin 

Rac1 

ub 

Dvl 

PAR-1 

ub TAB2 

TAK1 

TAB1 

NLK 

Extracellular 

Space 

Signal 

Receiving 

Cell 

Notch 

Fringe 

Deltex 

NEDD4 

Golgi 

ub 

Notch 

Recycling 

Activated 

Receptor 

Endosome 

NUMB 

α-Adaptin 

ADAM/ 

TACE 

S2 Cleavage 

TM 

NICD 

S3 Cleavage 

FBX7 

Nicastrin 

Presenilin 

APH-1 

PEN-2 

γ-Secretase 

Complex 

Ubiquitin 

Degradation 

Cytoplasm 

Nucleus 

OFF 

HDAC 

Groucho 

TCF3 

HDAC 

Groucho β-catenin 

Hesx1 

FoxO 

Pygo BCL9 

Brg1 

β-catenin 

CBP 

LEF1/TCF1 

FoxO 

ICAT 

β-catenin 

Chibby 

NLK 

TAZ 

Target Genes: 

Myc, Cyclin D1, 

TCF-1, PPAR-δ, 

MMP-7, Axin-2, 

CD44, etc. 

Pit1 

Snail1 

Migration 

Adhesion 

S1 Cleavage Furin 

ER 

Cytoplasm 

Nucleus 

O-Fut 

CtBP1 CIR HDAC 

KDM5A 

SMRT SHARP 

CSL SKIP 

Inactive 

Lysosomal 

Degradation 

KDM5A 

HAT 

MAML 

SKIP 

CSL 

Active 

Notch Target Genes 

HES Family 

Myc 

p21 

Cyclin D3 

The conserved Wnt/β-Catenin pathway regulates stem cell pluripotency and cell fate decisions during development. This developmental cascade integrates signals from other 

pathways, including retinoic acid, FGF, TGF-β, and BMP, within different cell types and tissues. The Wnt ligand is a secreted glycoprotein that binds to Frizzled receptors, leading 

to the formation of a larger cell surface complex with LRP5/6. Frizzleds are ubiquitinated by ZNRF3 and RNF43, whose activity is inhibited by R-spondin binding to LGR5/6. 

In this manner R-spondins increase sensitivity of cells to the Wnt ligand. Activation of the Wnt receptor complex triggers displacement of the multifunctional kinase GSK-3β 

from a regulatory APC/Axin/GSK-3β-complex. In the absence of Wnt-signal (Off-state), β-catenin, an integral E-cadherin cell-cell adhesion adaptor protein and transcriptional 

co-regulator, is targeted by coordinated phosphorylation by CK1 and the APC/Axin/GSK-3β-complex leading to its ubiquitination and proteasomal degradation through the 

β-TrCP/Skp pathway. In the presence of Wnt ligand (On-state), the co-receptor LRP5/6 is brought in complex with Wnt-bound Frizzled. This leads to activation of Dishevelled 

(Dvl) by sequential phosphorylation, poly-ubiquitination, and polymerization, which displaces GSK-3β from APC/Axin through an unclear mechanism that may involve substrate 

trapping and/ or endosome sequestration. Stablized β-catenin is translocated to the nucleus via Rac1 and other factors, where it binds to LEF/TCF transcription factors, displacing 

co-repressors and recruiting additional co-activators to Wnt target genes. Additionally, β-catenin cooperates with several other transcription factors to regulate specific 

targets. Importantly, researchers have found β-catenin point mutations in human tumors that prevent GSK-3β phosphorylation and thus lead to its aberrant accumulation. 

E-cadherin, APC, R-spondin and Axin mutations have also been documented in tumor samples, underscoring the deregulation of this pathway in cancer. Wnt signaling has 

also been shown to promote nuclear accumulation of other transcriptional regulator implicated in cancer, such as TAZ and Snail1. Furthermore, GSK-3β is involved in glycogen 

metabolism and other signaling pathways, which has made its inhibition relevant to diabetes and neurodegenerative disorders. 


Angers, S. and Moon, R.T. (2009) Nat. Rev. Mol. Cell Biol. 10, 468–477. • Cadigan, K.M. and Waterman, M.L. (2012) Cold Spring Harb. Perspect. Biol. 4, a007906. • 

Clevers, H. and Nusse, R. (2012) Cell 149, 1192–1205. • de Lau, W., Peng, W.C., Gros, P. and Clevers, H. (2014) Genes Dev. 28, 305–316. • Fearon, E.R. (2009) Cancer 

Cell 16, 366–368. • MacDonald, B.T., Tamai, K. and He, X. (2009) Dev. Cell 17, 9–26. • Metcalfe, C. and Bienz, M. (2011) J. Cell. Sci. 124, 3537–3544. • Niehrs, C. 

(2012) Nat. Rev. Mol. Cell Biol. 13, 767–779. • Nusse, R. (2010) The Wnt Homepage. • Petersen, C.P. and Reddien, P.W. (2009) Cell 139, 1056–1068. • Sokol, S.Y. 

(2011) Development 138, 4341–4350. • van Amerongen, R. and Nusse, R. (2009) Development 136, 3205–3214. • Valenta, T., Hausmann, G., and Basler, K. (2012) 

EMBO J. 31, 2714–2736. 

© 2003–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Kenneth Cadigan, University of Michigan, Ann Arbor, MI, for reviewing this diagram. 


Notch signaling is an evolutionarily conserved pathway in multicellular organisms that regulates cell-fate determination during development and maintains adult tissue homeostasis. 

The Notch pathway mediates juxtacrine cellular signaling wherein both the signal sending and receiving cells are affected through ligand-receptor crosstalk by which 

an array of cell fate decisions in neuronal, cardiac, immune, and endocrine development are regulated. Notch receptors are single-pass transmembrane proteins composed 

of functional extracellular (NECD), transmembrane (TM), and intracellular (NICD) domains. Notch receptors are processed in the ER and Golgi within the signal-receiving cell 

through cleavage and glycosylation, generating a Ca 2+ -stabilized heterodimer composed of NECD noncovalently attached to the TM-NICD inserted in the membrane (S1 

cleavage). The processed receptor is then endosome-transported to the plasma membrane to enable ligand binding in a manner regulated by Deltex and inhibited by NUMB. 

In mammalian signal-sending cells, members of the Delta-like (DLL1, DLL3, DLL4) and the Jagged (JAG1, JAG2) families serve as ligands for Notch signaling receptors. 

Upon ligand binding, the NECD is cleaved away (S2 cleavage) from the TM-NICD domain by TACE (TNF-α ADAM metalloprotease converting enzyme). The NECD remains 

bound to the ligand and this complex undergoes endocytosis/recycling within the signal-sending cell in a manner dependent on ubiquitination by Mib. In the signal-receiving 

cell, γ-secretase (also involved in Alzheimer’s disease) releases the NICD from the TM (S3 cleavage), which allows for nuclear translocation where it associates with the CSL 

(CBF1/Su(H)/Lag-1) transcription factor complex, resulting in subsequent activation of the canonical Notch target genes: Myc, p21, and the HES-family members. The Notch 

signaling pathway has spurred interest for pharmacological intervention due to its connection to human disease. Importantly, researchers have found Notch receptor activating 

mutations leading to nuclear accumulation of NICD are common in adult T cell acute lymphoblastic leukemia and lymphoma. In addition, loss-of-function Notch receptor and 

ligand mutations are implicated in several disorders, including Alagille syndrome and CADASIL, an autosomal dominant form of cerebral arteriopathy. 


Ables, J.L., Breunig, J.J., Eisch, A.J., Rakic, P. (2011) Nat. Rev. Neurosci. 12, 269–283. • Andersson, E.R., Lendahl, U. (2014) Nat. Rev. Drug Discov. 13, 357–378. • Aster, 

J.C., Blacklow, S.C., Pear, W.S. (2011) J. Pathol. 223, 262–273. • Bai, G., Pfaff, S.L. (2011) Neuron 72, 9–21. • de la Pompa, J.L., Epstein, J.A. (2012) Dev. Cell 22, 244– 

254. • Ntziachristos, P., Lim, J.S., Sage, J., Aifantis, I. (2014) Cancer Cell. 24. • Ranganathan, P., Weaver, K.L., Capobianco, A.J. (2011) Nat. Rev. Cancer 11, 338–351. 

• Weinmaster, G., Fischer, J.A. (2011) Dev. Cell 21, 134–144. • Yuan, J.S., Kousis, P.C., Suliman, S., Visan, I., Guidos, C.J. (2010) Annu. Rev. Immunol. 28, 343–365. 





Hippo Signaling 

Adherens Junction Tight Junction 

Crb 

ZO-2 

YAP/TAZ 

AMOT 

YAP/TAZ 

AMOT 

β-TrCP 

FRMD 

Merlin 

KIBRA 

SIK1-3 

α-catenin 

Mst1/2 

LKB1 

14-3-3 SAV1 

MARK4 

YAP 

14-3-3 

YAP 

RASSF 

Ajuba 

CK1 

Merlin 

CD44 

 

FAT4 

 

LATS1/2 

YAP/TAZ 

 

TAOK-1-3 

MOB1 


PP2A 

F-actin 

ITCH 

PP1 

Gα 12/13 

ASPP2 

Gα q/11 

Gα i/o 

AMOT 

Gα s 

Mechanical Signaling 

Rho 

Hedgehog Signaling 

PKA 

SUFU 

GLI 1/2/3 

SUFU 

β−TrCP 

CK1 

GLI 1/2/3 

GSK-3β 

Proteasome 

Repressor GLI 3-R 

no transcription of 

target genes 

Cytoplasm 

KIF7 

Primary 

Cilium 

GLI 1/2/3 

Microtubules 

SMO 

GLI 1/2 Degradation 

Off-State 

Skn 

PTCH1 

GLI 3-R 

SCUBE2 

CDON/BOC 

Nucleus 

Hh 

HhN 

DISP1 

PTCH2 

Primary 

Cilium 

ER/Golgi 

HhN 

Microtubules 

SUFU 

GLI 1/2/3 

GLI 1/2-Act 

Activator 

KCTD11 

Cyclin D, Cyclin E, 

Myc, GLI1, PTCH1, 

PTCH2, Hhip1 

Cytoplasm 

KIF7 

Hh Secreting Cell 

Gα i 

β-Arrestin 

SMO 

On-State 

PTCH1 

GLI 1/2-Act 

Mammalian 

Ligands: 

Ihh 

Dhh 

Shh 

CDON/BOC 

Nucleus 

Hhip1 GAS1 

The evolutionarily conserved Hedgehog (Hh) pathway is essential for normal embryonic development and plays critical roles in adult tissue maintenance, renewal and regeneration. 

Secreted Hh proteins act in a concentration- and time-dependent manner to initiate a series of cellular responses that range from survival and proliferation to cell fate 

specification and differentiation. 

HhN 

VGL4 

TEAD1-4 

WBP2 

YAP/TAZ 

TEAD1-4 

YAP/TAZ 

Smad 

YAP/TAZ 

p73 

YAP/TAZ 

RUNX 

Nucleus 

Transcription 

Cytoplasm 

Hippo signaling is an evolutionarily conserved pathway that controls organ size by regulating cell proliferation, apoptosis, and stem cell self renewal. In addition, dysregulation 

of the Hippo pathway contributes to cancer development. Core to the Hippo pathway is a kinase cascade, wherein Mst1/2 (ortholog of Drosophila Hippo) kinases and SAV1 

form a complex to phosphorylate and activate LATS1/2. LATS1/2 kinases in turn phosphorylate and inhibit the transcription co-activators YAP and TAZ, two major downstream 

effectors of the Hippo pathway. When dephosphorylated, YAP/TAZ translocate into the nucleus and interact with TEAD1-4 and other transcription factors to induce expression 

of genes that promote cell proliferation and inhibit apoptosis. The Hippo pathway is involved in cell contact inhibition, and its activity is regulated at multiple levels: Mst1/2 and 

LATS1/2 are regulated by upstream molecules such as Merlin, KIBRA, RASSFs, and Ajuba; 14-3-3, α-catenin, AMOT, and ZO-2 retain YAP/TAZ in the cytoplasm, adherens 

junctions, or tight junctions by binding; Mst1/2 and YAP/TAZ phosphorylation and activity are modulated by phosphatases; Lats1/2 and YAP/TAZ stability are regulated by 

protein ubiquitination; and LATS1/2 activity is also regulated by the cytoskeleton. Despite extensive study of the Hippo pathway in the past decade, the exact nature of extracellular 

signals and membrane receptors regulating the Hippo pathway remains elusive. 


Badouel, C. and McNeill, H. (2011) Cell 145, 484–484. • Genevet, A. and Tapon, N. (2011) Biochem. J. 436, 213–224. • O’Hayre, M., Degese, M.S., and Gutkind, J.S. 

(2014) Curr. Opin. Cell Biol. 27, 126–135. • Pan, D. (2010) Dev. Cell 19, 491–505. • Sudol, M. and Harvey, K.F. (2010) Trends Biochem. Sci. 35, 627–633. • Yu, F.X. 

and Guan, K.L. (2013) Genes Dev. 27, 355–371. • Zhao, B., Li, L., Lei, Q., and Guan, K.L. (2010) Genes Dev. 24, 862–874. • Zhao, B., Tumaneng, K., and Guan, K.L. 

(2011) Nat. Cell Biol. 13, 877–883. 

Proper levels of Hh signaling require the regulated production, processing, secretion and trafficking of Hh ligands– in mammals this includes Sonic (Shh), Indian (Ihh) and 

Desert (Dhh). All Hh ligands are synthesized as precursor proteins that undergo autocatalytic cleavage and concomitant cholesterol modification at the carboxy terminus 

and palmitoylation at the amino terminus, resulting in a secreted, dually-lipidated protein. Hh ligands are released from the cell surface through the combined actions of 

Dispatched and Scube2, and subsequently trafficked over multiple cells through interactions with the cell surface proteins LRP2 and the Glypican family of heparan sulfate 

proteoglycans (GPC1-6). 

Hh proteins initiate signaling through binding to the canonical receptor Patched (PTCH1) and to the co-receptors GAS1, CDON and BOC. Hh binding to PTCH1 results in derepression 

of the GPCR-like protein Smoothened (SMO) that results in SMO accumulation in cilia and phosphorylation of its cytoplasmic tail. SMO mediates downstream signal 

transduction that includes dissociation of GLI proteins (the transcriptional effectors of the Hh pathway) from kinesin-family protein, Kif7, and the key intracellular Hh pathway 

regulator SUFU. 

GLI proteins also traffic through cilia and in the absence of Hh signaling are sequestered by SUFU and Kif7, allowing for GLI phosphorylation by PKA, GSK3β and CK1, and 

subsequent processing into transcriptional repressors (through cleavage of the carboxy-terminus) or targeting for degradation (mediated by the E3 ubiquitin ligase β-TrCP). In 

response to activation of Hh signaling, GLI proteins are differentially phopshorylated and processed into transcriptional activators that induce expression of Hh target genes, 

many of which are components of the pathway (e.g. PTCH1 and GLI1). Feedback mechanisms include the induction of Hh pathway antagonists (PTCH1, PTCH2 and Hhip1) 

that interfere with Hh ligand function, and GLI protein degradation mediated by the E3 ubiquitin ligase adaptor protein, SPOP. 

In addition to vital roles during normal embryonic development and adult tissue homeostasis, aberrant Hh signaling is responsible for the initiation of a growing number of 

cancers including, classically, basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma; more recently overactive Hh signaling has been implicated in pancreatic, 

lung, prostate, ovarian, and breast cancer. Thus, understanding the mechanisms that control Hh pathway activity will inform the development of novel therapeutics to treat a 

growing number of Hh-driven pathologies. 


Beachy, P.A., Hymowitz, S.G., Lazarus, R.A., Leahy, D.J., and Siebold, C. (2010) Genes Dev. 24, 2001–2012. • Eaton, S. (2008) Nat. Rev. Mol. Cell Biol. 9, 437–445. • 

Hui, C.C. and Angers, S. (2011) Annu. Rev. Cell Dev. Biol. 27, 513–537. • Ingham, P.W., Nakano, Y., and Seger, C. (2011) Rev. Genet. 12, 393–406. • Ng, J.M. and 

Curran, T. (2011) Nat. Rev. Cancer 11, 493–501. • Wilson, C.W. and Chuang, P.T. (2010) Development 137, 2079–2094. • Teglund, S., and Toftgard, R. (2010) Biochim. 

Biophys. Acta. 1805, 181–208. • Briscoe, J., and Therond, P.P. (2013) Nat. Rev. Mol. Cell Biol. 14, 416–429. • Goetz, S.C., and Anderson, K.V. (2010) Nat. Rev. Genet. 

11, 331–344. • Falkenstein, K.N., and Vokes, S.A. (2014) Semin. Cell Dev. Biol. 33, 73–80. 

© 2010–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Kun-Liang Guan, University of California, San Diego, CA for reviewing this diagram. 


© 2006–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Hans Widlund, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, for reviewing this diagram. 




TGF-b Signaling 

ESC Pluripotency Differentiation 

RhoA 

mDia ROCK 

MLC LIMK 

Cofilin 

PAK 

Rac/ 

Cdc42 

c-Abl 

TGF-β Ligands: 

TGF-βs, Activins, Nodals 

Par6 

PKC 

PI3K 

Akt 

mTOR 

Type II 

Receptor 

PP2A 

Type I 

Receptor 

Shc 

SARA 

Smad2/3 

TRAF4/6 

Smurf2 Smad7 

JNK 

GRB2 

USP4/11/15 

TAK1 

p38 

NF-κB 

SOS 

TMEPAI 

Smad7 

Smad2/3 

Smurf2 

Smad2/3 Smad4 

Smad2/3 

Ras 

Erk1/2 

Smad4 

Smurf1 

Type I 

Receptor 

Smad1/5/9 

Smad1/5/9 

Smad1/5/9 Smad4 

Smad1/5/9 

BMP Ligands: 

BMP-2, -4, -7, MIS 

Smurf1 

Smad6/7 

Type II 

Receptor 

LIMK 

Cofilin 

p38 

TAK1 

JNK 

IGF-IR 

PI3K 

Akt 

FGFR 

Ras/ 

Raf 

MEK1/2 

Erk1/2 

LRP 

Wnt 

Frizzled 

Gα q/o 

PP2A 

APC 

Axin GSK-3 

β-catenin 

β-catenin 

TGF-β/Activin/Nodal 

SARA 

Smad2/3 

Smad2/3 

Smad2/3 Smad4 

Smad2/3 

Smad4 

BMP 

Smad1/5/9 

Smad1/5/9 

γ−Secretase 

Smad1/5/9 Smad4 

Smad1/5/9 

Notch 

NICD 

Actin Polymerization 

Stress Fibers 

4E- 

BP1/2 

Cell Adhesion 

p70 S6K 

Actin Polymerization 

Stress Fibers 

Cytoplasm 

Nucleus 

Erk1/2 

TCFs 

β-catenin 

Smad2/3 

Smad1/5/9 

NICD 

Cytoplasm 

Nucleus 

Smad4 CBP 

Smad2/3 

Smad2/3 TF 

Smad4 CBP 

Smad1/5/9 

Smad1/5/9 TF 

Gene Expression 

Oct-4 Sox2 Nanog 



Polycomb 

Transcription Factors: 

AP-1 homeodomain 

bZIP Sp1 

RUNX nuclear receptors 

Fox IRF-7 

bHLH 

Corepressors: 

c-Ski/SnoN 

Evi1 

TGIF 

SIP1 

Tob (BMP only) 

HDAC4/5 

Coactivators: 

CBP/p300 

SMIF 

MSG1 

ARC105 

Pluripotency and Self Renewal 

hESC markers: 

Oct-4 SSEA3/4 

Sox2 TRA-1-60 

Nanog TRA-1-81 


Endoderm 

Mesoderm 

Ectoderm 

Primordial Germ Cells 

Transforming growth factor-β (TGF-β) superfamily signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological 

systems. In general, signaling is initiated with ligand-induced oligomerization of serine/threonine receptor kinases and phosphorylation of the cytoplasmic signaling molecules 

Smad2 and Smad3 for the TGF-β/activin pathway, or Smad1/5/9 for the bone morphogenetic protein (BMP) pathway. Carboxy-terminal phosphorylation of Smads by activated 

receptors results in their partnering with the common signaling transducer Smad4, and translocation to the nucleus. Activated Smads regulate diverse biological effects by 

partnering with transcription factors resulting in cell-state specific modulation of transcription. Inhibitory Smads, i.e. Smad6 and Smad7 antagonize activation of R-Smads. 

The expression of inhibitory Smads (I-Smads) 6 and 7 is induced by both activin/TGF-β and BMP signaling as part of a negative feedback loop. The stability of TGF-β family 

receptors and/or Smads are regulated by Smurf E3 ubiquitin ligases and USP4/11/15 deubiquitinases. TGF-β/activin and BMP pathways are modulated by MAPK signaling 

at a number of levels. Moreover, in certain contexts, TGF-β signaling can also affect Smad-independent pathways, including Erk, SAPK/JNK, and p38 MAPK pathways. Rho 

GTPase (RhoA) activates downstream target proteins, such as mDia and ROCK, to prompt rearrangement of the cytoskeletal elements associated with cell spreading, cell 

growth regulation, and cytokinesis. Cdc42/Rac regulates cell adhesion through downstream effector kinases PAK, PKC, and c-Abl following TGF-β activation. 


Horbelt, D., Denkis, A., and Knaus, P. (2012) Int. J. Biochem. Cell Biol. 44, 469–474. • Ikushima, H. and Miyazono, K. (2010) Nat. Rev. Cancer 10, 415–424. • Kitisin, K., 

Saha, T., Blake, T., et al. (2007) Sci. STKE 399, cm1. • Meulmeester, E. and ten Dijke, P. (2011) J. Pathol. 223, 205–218. • Schmierer, B. and Hill, C.S. (2007) Nat. Rev. 

Mol. Cell Biol. 8, 970–982. • Xiao, Y.T., Xiang, L.X., and Shao, J.Z. (2007) Biochem. Biophys. Res. Commun. 362, 550–553. • Zhang, L., Zhou, F., de Vinuesa, A.G., et al. 

(2013) Mol. Cell 51, 559–572. 

Two distinguishing characteristics of embryonic stem cells (ESCs) are pluripotency and the ability to self-renew. These traits, which allow ESCs to grow into any cell type in 

the adult body and divide continuously in the undifferentiated state, are regulated by a number of cell signaling pathways. In human ESCs (hESCs), the predominant signaling 

pathways involved in pluripotency and self-renewal are TGF-β, which signals through Smad2/3/4, and FGFR, which activates the MAPK and Akt pathways. The Wnt pathway 

also promotes pluripotency, although this may occur through a non-canonical mechanism involving a balance between the transcriptional activator, TCF1, and the repressor, 

TCF3. Signaling through these pathways supports the pluripotent state, which relies predominantly upon three key transcription factors: Oct-4, Sox2, and Nanog. These 

transcription factors activate gene expression of ESC-specific genes, regulate their own expression, suppress genes involved in differentiation, and also serve as hESCs markers. 

Other markers used to identify hESCs are the cell surface glycolipid SSEA3/4, and glycoproteins TRA-1-60 and TRA-1-81. In vitro, hESCs can be coaxed into derivatives of the 

three primary germ layers, endoderm, mesoderm, or ectoderm, as well as primordial germ cell-like cells. One of the primary signaling pathways responsible for this process is 

the BMP pathway, which uses Smad1/5/9 to promote differentiation by both inhibiting expression of Nanog, as well as activating the expression of differentiation-specific genes. 

Notch also plays a role in differentiation through the notch intracellular domain (NICD). As differentiation continues, cells from each primary germ layer further differentiate along 

lineage-specific pathways. 


Bilic, J. and Izpisua Belmonte, J.C. (2012) Stem Cells 30, 33–41. • Dalton, S. (2013) Curr. Opin. Cell Biol. 25, 241–246. • Guenther, M.G. (2011) Epigenomics 3, 323–343. 

• Jaenisch, R. and Young, R. (2008) Cell 132, 567–582. • Ng, H.H. and Surani, M.A. (2011) Nat. Cell Biol. 13, 490–496. • Pan, G. and Thomson, J.A. (2007) Cell Res. 

17, 42–49. • Welham, M.J., Kingham, E., Sanchez-Ripoll, Y., Kumpfmueller, B., Storm, M., and Bone, H. (2011) Biochem. Soc. Trans. 39, 674–678. • Young, R.A. (2011) 

Cell 144, 940–954. 

© 2003–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Peter ten Dijke, Leiden University Medical Center, Leiden, The Netherlands, for reviewing this diagram. 


© 2009–2015 Cell Signaling Technology, Inc. • We would like to thank Justin Brumbaugh and Prof. Konrad Hochedlinger, 

HHMI and MGH Cancer Center, Center for Regenerative Medicine, Harvard University, Cambridge, MA, for reviewing this diagram. 



Molecular rendering of 

tumor vascularization 

Angiogenesis 

Angiogenesis is the formation of new blood vessels from pre-existing larger blood vessels. It is required 

during embryogenesis, and defects in or interference with this process have severe consequences 

for embryonic development. In adult organisms, angiogenesis plays a key role in normal physiological 

processes such as wound healing and inflammation, and in mammalian placental development. 

The process of angiogenesis can also be hijacked by tumors, which require a supply of oxygen and 

nutrients to support the high metabolic rate of tumor cells. 

VEGF Receptor Signaling 

Vascular endothelial growth factor receptors (VEGFR) are a receptor tyrosine kinase family composed of 

seven extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail 

containing an active kinase domain. VEGFR1 plays an important role in EC function and normal vascular 

development, as well as in hematopoietic function. VEGFR2 is a major receptor for VEGF-induced signaling 

in endothelial cells. Upon ligand binding, VEGFR2 undergoes autophosphorylation and becomes 

activated. Signaling from VEGFR2 is necessary for the execution of VEGF-stimulated proliferation, 

chemotaxis, and sprouting, as well as survival of cultured ECs in vitro and in vivo. VEGFR3 expression is 

largely restricted to adult lymphatic endothelium and is thought to control lymphangiogenesis. 

VEGFR phosphorylation 

at tyrosine residues 

activates downstream 

signaling cascades that 

support angiogenesis. 

Tyr801 

Tyr951 

Tyr966 

Tyr1008 

Tyr1054 

Tyr1059 

Tyr1175 

Tyr1214 

VEGF 

lg-like 

domains 

P 

Akt 

P TSAd 

P 

P PLCγ 

P 

Cbl 

P 

P PLCγ Cbl 

P Shb 

P Grb2 Gab1 

SHP2 

Src 

PI3K 

PLCγ 

PI3K 

Src 


Vascular permeability, 


Endothelial cell migration 

Differentiation, tubulogenesis 

Cell proliferation, angiogenesis 

Focal adhesion formation, 


Cell migration, lamellipodia 

formation, capillary formation 

Kinase domain 

Autophosphorylation 

Angiogenesis Signaling 

When angiogenesis is stimulated, pro-angiogenic growth factors such as VEGF, PDGF, FGF, and TGF 

are released. These growth factors bind their cognate receptors on endothelial cells (EC) within preexisting 

vessels. This triggers a signaling cascade that activates several signaling pathways such as 

PI3K/Akt, Erk1/2, Smad, and Notch and results in EC proliferation and migration. New blood vessel 

formation occurs as ECs use matrix metalloproteases (MMPs) and integrins to digest extracellular 

matrix and migrate into new territory where they lengthen and form tubes. 

VEGF treatment results in phosphorylation 

of VEGFR-2 at Tyr1175. 

PathScan ® Phospho-VEGFR-2 (Tyr1175) Sandwich ELISA Kit #7335: 

Treatment of HUVEC with VEGF stimulates phosphorylation of VEGFR-2 at 

Tyr1175, detected by #7335, but does not affect the level of total VEGFR-2 

protein detected by PathScan ® Total VEGFR-2 Sandwich ELISA kit #7340. 

Absorbance at 450 nm is shown in the top figure, while the corresponding 

western blot using Phospho-VEGFR-2 (Tyr1175) Rabbit mAb #2478 (right 

panel) or VEGFR-2 Rabbit mAb (7340-55B11) (left panel), is shown in the 

bottom figure. 

Untreated 

VEGF-treated 

Absorbance 450nm 

3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0 

kDa 

200 

140 

Total VEGFR-2 

Phospho-VEGFR-2 

(Tyr1175) 

– + – + VEGF 

TPA induces expression of MMP-9, a protease that digests 

extracellular matrix and is associated with tumor angiogenesis. 

MMP-9 (D6O3H) XP ® Rabbit mAb #13667: 

IHC analysis of paraffin-embedded human breast 

carcinoma (A) using #13667. WB analysis of 

concentrated, serum-free cultured medium from 

U-2 OS cells, untreated (-) or treated with TPA 

#4174 (200 nM, 48 hr; +) (B), using #13667. 

A 

B 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

– + 

MMP-9 

TPA 

Neuropilin-2 

In addition to neuronal guidance, the single pass, transmembrane glycoprotein neuropilin-2 

is a co-receptor for VEGFR2 and VEGFR3, and plays a role in the development of vascular 

and lymphatic systems through binding VEGF 165 and VEGF145. 

Neuropilin-2 is expressed in the 

developing mouse embryo. 

Neuropilin-2 (D39A5) XP ® Rabbit mAb #3366: Confocal 

IF analysis of E14.5 mouse embryo using #3366 (green). 


CST Technical 

Support 

When you contact CST for technical 

support, you can be confident you 

will be working with a colleague 

you can trust, striving to provide the 

highest quality products and services 

to you, our customer. Please see our 

Technical Support resource page 

online for contact information. 

www.cellsignal.com/cstsupport 


www.cellsignal.com/cstangiogenesis 

167



Elevated PDGF 

Receptor β expression 

is found within stromal 

cells of many forms 

of cancer. 

PDGF Receptor β (28E1) Rabbit mAb 


human colon carcinoma using #3169. 

CD73 expression on 

tumor cells promotes 

angiogenesis. 

NT5E/CD73 (D7F9A) Rabbit mAb 



Pericyte Signaling 

Pericytes are support cells that provide structural stability for newly formed blood vessels, promote EC 

survival, and regulate vasoconstriction and dilation. This is done through a reciprocal signaling mechanism 

in which PDGF-BB secreted into the matrix by ECs acts as a ligand for PDGFR-β located on the 

pericyte membrane. In return, pericytes produce and secrete VEGF that signals through the endothelial 

VEGF receptor. 

Tumor Signaling 

Tumor angiogenesis occurs when cancer cells stimulate new blood vessel growth in order to bring 

oxygen and nutrients to a tumor. As a tumor grows in size, diffusion is no longer sufficient to oxygenate 

the cells at the center of the mass, creating a hypoxic environment. Hypoxia stabilizes levels of HIF-1α, 

a transcription factor that responds to changing oxygen levels. Under hypoxic conditions, HIF-1α binds 

HIF-1β to activate transcription of angiogenesis-promoting genes. Cancer cells also secrete a variety 

of growth factors and cytokines that stimulate classical angiogenic signaling pathways, extracellular 

matrix remodeling, and an inflammatory response that leads to new blood vessel formation. 

Under normoxia conditions, HIF-1α is 

hydroxylated at Pro564, marking it for degradation. 

PathScan ® Hydroxy-HIF-1α (Pro564) Sandwich ELISA Kit #13201: 

Treatment of HeLa cells with the hydroxylase inhibitor dimethyloxaloylglycine 

(DMOG) results in decreased hydroxylation of HIF-1α, as detected by 

#13201, but does not affect the level of total HIF-1α detected by PathScan ® 

Total HIF-1α Sandwich ELISA Kit #13127. Absorbance at 450 nm is shown 

in the top figures, while corresponding western blots using a total HIF-1α 

antibody (left) and Hydroxy-HIF-1α (Pro564) (D43B5) XP ® Rabbit mAb #3434 

(right) are shown in the bottom figures. Treatment of HeLa cells with the 

proteasome inhibitor MG-132 #2194 stabilizes HIF-1α protein. 

Untreated 

MG-132-treated 

MG-132-treated, 

DMOG-treated 

Total HIF-1α 

Hydroxy-HIF-1α 

(Pro564) 

Angiogenesis in Cancer 

Cancer cells secrete a variety of growth factors and cytokines that stimulate classical angiogenesis 

signaling pathways and extracellular matrix remodeling. Cytokines can also induce an inflammatory 

response that initiates angiogenesis and consequent vascularization of the tumor. Sprouting angiogenesis 

is the first step in this neovascularization process. In response to stimuli released by tumor cells, 

a single EC migrates toward the angiogenic factors and proliferates to form sprouts of ECs surrounding 

a lumen that is connected to the mother blood vessel. Tumors may also be vascularized by intussusceptive 

angiogenesis. This process proceeds much more quickly than sprouting and is essentially the 

splitting of an existing blood vessel into two new vessels. The ECs, pericytes, and basement membrane 

associated with a tumor exhibit abnormalities leading to tortuous, poorly organized, and leaky vasculature. 

However, this sub-optimal structure is often sufficient to supply the tumor with oxygen, nutrients, 

and soluble factors that help it survive and expand. It is through this vascular system that some tumor 

cells are able to escape and travel through the circulatory system to new locations. Such metastatic 

colonization is a key factor in the fatal outcomes of many types of cancer, and the density of tumor 

angiogenesis has been linked to tumor metastasis and patient survival rates. Accordingly, angiogenesis 

is under investigation as an important prognostic indicator in cancer, and angiogenesis inhibitors are 

attractive as candidate therapeutics. 


3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0 

– + + – + + MG-132 

– – + – – + DMOG 

Angiogenesis Resources 

Please visit our website for additional resources and products relating to the study of Angiogenesis. 


In ECs, VE-Cadherin signaling, expression, and localization 

correlate with vascular permeability and tumor angiogenesis. 

VE-Cadherin (D87F2) XP ® Rabbit 


HUVEC (VE-Cadherin positive; left) and 

HeLa cells (VE-Cadherin negative; right) 


have been labeled with DY-554 phalloidin 



FGFR1 binds acidic FGF and basic FGF, both potent promoters of angiogenesis. 

A 

B 

Events 

60 

50 

40 

60 

50 

40 

30 

FGF Receptor 1 

FGF Receptor 1 (D8E4) XP ® Rabbit mAb #9740: IHC analysis of paraffin-embedded human breast carcinoma (A) using 

#9740. Flow cytometric analysis of A-204 cells (B) using #9740 (blue) compared to concentration matched Rabbit (DA1E) 

mAb IgG XP ® Isotype Control #3900 (red). WB analysis of extracts from A-204 (FGFR1 positive), KG-1a (FGFR1 oncogenic 

partner-FGFR1 fusion), A172 (FGFR1 low expression), and HT-29 (FGFR1 negative) cells (C) using #9740 (upper) and β-Actin 

(D6A8) Rabbit mAb #8457 (lower). 

Angiogenesis Modulators 


Benazzi, C., Al-Dissi, A., Chau, C.H., et al. (2014) Scientific World Journal 2014, 919570. • Claesson-Welsh, L. (2003) 

Biochem. Soc. Trans. 31, 20–24. • Ferrara, N., Gerber, H.P., and LeCouter, J. (2003) Nat. Med. 9, 699–676. • Jain, R.K. 

(2005) Science 307, 58–62. • Karkkainen, M.J. and Petrova, T.V. (2000) Oncogene 19, 5598–5605. • Koch, S., Tugues, S., 

Li, X., Gualandi, L., and Claesson-Welsh, L. (2011) Biochem. J. 437, 169–183. • Lu, X. and Kang, Y. (2010) Clin. Cancer Res. 

16, 5928–5935. • Makanya, A.N., Hluchchuk, R., and Djonov, V.G. (2009) Angiogenesis 12, 113–123. • Meyer, M., Clauss, 

M., Lepple-Wienhues, A., et al. (1999) EMBO J. 18, 363–374. • Patel-Hett, S. and D’Amore, P.A. (2011) Int. J. Dev. Biol. 55, 

353–363. • Rahimi, N., Dayanir, V., and Lashkari, K. (2000) J. Biol. Chem. 275, 16986–16992. • Ribatti, D., Nico, B., and 

Crivellato, E. (2011) Int. J. Dev. Biol. 55, 261–268. • Robinson, C.J. and Stringer, S.E. (2001) J. Cell Sci. 114, 853–865. • 

Saharinen, P., Eklund, L., Pulkki, K., et al. (2011) Trends Mol. Med. 17, 347–362. • Sakurai, T. and Kudo, M. (2011) Oncology 

81 Suppl 1, 24–29. • Senger, D.R. and Davis, G.E. (2011) Cold Spring Harb. Perspect Biol. 3, a005090. • Weis, S.M. and 

Cheresh, D.A. (2011) Nat. Med. 17, 1359–1370. 

C 

kDa 

200 

140 

100 

80 

1 2 3 4 

Promoters of Angiogenesis 

Inhibitors of Angiogenesis 

Acidic fibroblast growth factor (aFGF) #5234 

ADAMTS1 

Angiogenin 

Angiostatin 

Basic fibroblast growth factor (bFGF) #8910 

Endostatin 

Epidermal growth factor (EGF) #8916 Interferons (alpha) #8927 

Granulocyte colony-stimulating factor (GM-CSF) #8922 Platelet factor 4 

Hepatocyte growth factor (HGF) 

Prolactin 16 kDa fragment 

Interleukin 8 (IL-8) #8921 

Thrombospondin 

Placental growth factor (PGF) 

Tissue inhibitor of metalloproteinase-1 (TIMP-1) 

Platelet-derived endothelial growth factor (PEGF) 


Transforming growth factor alpha (TGF-α) #5495 


Tumor necrosis factor alpha (TNF-α) #8902 

Vascular endothelial growth factor (VEGF) #8908, #8065 

FGFR1 

FOP- 

FGFR1 

β-Actin 

Lanes 

1. A-204 

2. KG-1a 

3. A172 

4. HT-29 



169



nodes within angiogenesis signaling 


in angiogenesis research. Primary 











163 


CST antibodies for VEGF Receptor 2 

have been cited over 163 times in highimpact, 

peer-reviewed publications from 

the global research community. 

Commonly Studied Angiogenesis Targets 


ADAMTS1 

• 

Angiopoietin-2 

• 

CA9 

• 

CBP 

• • 

Acetyl-CBP (Lys1535)/p300 (Lys1499) • 

CD31 (PECAM-1) 

• 

Cripto 

• • 

CYR61 

• 

DLL4 

• 

eNOS 

• • 

EphA2 

• • 


EphB1 

• 

Acidic FGF 

• 

FGF Receptor 1 • • • • 


• • • • 


• • 

FGF Receptor 4 • • • 

FIH 

• 

Gremlin 

• 

HIF-1α 

• • 

Hydroxy-HIF-1α (Pro564) • • 

HIF-1β/ARNT 

• • 

HO-1 

• • 

Integrin α6 

• 

Integrin αV 

• 

Integrin β1 

• • 

Integrin β3 

• • 

Integrin β5 

• • 

Jagged1 

• 

Maspin 

• 

MMP-2 

• • 

MMP-7 

• 

MMP-9 

• • 

NDRG1 • • • 


Whiteus, C. et al. (2014) Perturbed neural activity disrupts 

cerebral angiogenesis during a postnatal critical period. 

Nature 505, 407−411. 

Behjati, S. et al. (2014) Recurrent PTPRB and PLCG1 mutations 

in angiosarcoma. Nat. Genet. 46, 376−379. 

Chen, P.Y., Qin, L., Zhuang, Z.W., Tellides, G., Lax, I., 

Schlessinger, J., Simons, M. (2014) The docking protein 

FRS2alpha is a critical regulator of VEGF receptors signaling. 

Proc. Natl. Acad. Sci. USA 111, 5514−5519. 

Ria, R. et al. (2014) HIF-1alpha of bone marrow endothelial 

cells implies relapse and drug resistance in patients with 

multiple myeloma and may act as a therapeutic target. Clin. 

Cancer Res. 20, 847−858. 

Blosser, W. et al. (2014) A method to assess target gene involvement 

in angiogenesis in vitro and in vivo using lentiviral 

vectors expressing shRNA. PLoS One 9, e96036 

Riquelme, E. et al. (2014) VEGF/VEGFR-2 Upregulates EZH2 

Expression in Lung Adenocarcinoma Cells and EZH2 Depletion 

Enhances the Response to Platinum-Based and VEGFR- 

2-Targeted Therapy. Clin. Cancer Res. 20, 3849−3861. 

Yang, Y. et al. (2013) GAB2 induces tumor angiogenesis in 

NRAS-driven melanoma. Oncogene 32, 3627−3637. 

Nakayama, M. et al. (2013) Spatial regulation of VEGF receptor 

endocytosis in angiogenesis. Nat. Cell Biol. 15, 249−260. 


NDRG2 

• 

NDRG3 

• 

NDRG4 

• 

Neuropilin-2 

• 

Notch1 • • 

Cleaved Notch1 (Val1744) • • • 

Notch2 

• 

Notch3 

• • 

Notch4 

• 

NT5E/CD73 

• • 

PDGF Receptor α • • • • 

Phospho-PDGF Receptor α (Tyr754) • 

PDGF Receptor β • • • 

Phospho-PDGF Receptor β (Tyr740) • 



PHD-2/Egln1 

• • 

RECK 

• 

Ron 

• 

Phospho-Ron (panTyr) 

• 

Spry1 

• • 

Tenascin C 

• 

TGF-β Receptor I 

• 

TGF-β Receptor III 

• • 

Tie2 

• 

TIMP1 

• 

TIMP2 

• 

TIMP3 

• 

uPAR 

• • 

VE-Cadherin 

• • 

VEGF Receptor 1 

• 

VEGF Receptor 2 • • • • 

VEGF Receptor 3 

• • 

VHL 

• 

Planas-Paz, L. et al. (2012) Mechanoinduction of lymph 

vessel expansion. EMBO J. 31, 788−804. 

Fang, L. et al. (2013) Control of angiogenesis by AIBPmediated 

cholesterol efflux. Nature 498, 118−122. 

Harris, N.C. et al. (2013) The propeptides of VEGF-D 

determine heparin binding, receptor heterodimerization, and 

effects on tumor biology. J. Biol. Chem. 288, 8176−8186. 

Mujahid, S. et al. (2013) MiR-221 and miR-130a regulate 

lung airway and vascular development. PLoS One 8, 

e55911. 

Singh, N.K. et al. (2013) Both Kdr and Flt1 play a vital role in 

hypoxia-induced Src-PLD1-PKCgamma-cPLA(2) activation 

and retinal neovascularization. Blood 121, 1911−1923. 

Gourlaouen, M. et al. (2013) Essential role for endocytosis 

in the growth factor-stimulated activation of ERK1/2 in 

endothelial cells. J. Biol. Chem. 288, 7467−7480. 

Breitbach, C.J. et al. (2013) Oncolytic vaccinia virus disrupts 

tumor-associated vasculature in humans. Cancer Res. 73, 

1265−1275. 

Tang, J.R. et al. (2013) The NF-kappaB inhibitory proteins 

IkappaBalpha and IkappaBbeta mediate disparate responses 

to inflammation in fetal pulmonary endothelial cells. 

J. Immunol. 190, 2913−2923. 


Angiogenesis Signaling in Tumor Neovascularization 

Endothelial 

Cell 

Key 

Angiopoietin 1 

Angiopoietin 2 

bFGF 

Ephrin 

PDGF 

SLIT 

VEGF 

Tie2 

EPH 

ROBO3 

ROBO1/2 

Pericyte 

Integrins 

MMPs 

Basement 

Membrane 

Tip Cell 

FGFR 

Integrins 

VEGFR2 

Akt 

4E- 

BP1 

Tie2 

Neuropilin 

elF4E1 

• Growth Factors 

• Cytokines 

• ECM Proteases 

Tumor Cell 


PHDs 

Erk1/2 

HIF-1α 

Precursor 

Endothelial Cell 

OH 

HRE 

OH 

HIF-1α 

HIF-1α 

CBP/p300 

HIF-1β 

Normoxia 

HIF-1β 

Hypoxia 

Target Genes 

Platelet 

MMPs 

Nucleus 

PDGFR 

• Growth Factors 

• Cytokines 

• ECM Proteases 

Ets2 

HIF-1α 

Stat3 

Target Genes 

Tumor Associated 

Macrophage (TAM) 

Angiogenesis, the formation of new blood vessels from pre-existing blood vessels, plays a key role in tumorigenesis. When a small dormant tumor initiates angiogenesis, 

referred to as the ‘angiogenic switch’, it secretes factors that induce sprouting and chemotaxis of endothelial cells (ECs) towards the tumor mass. Within the hypoxic environment 

of the inner tumor mass the transcription factor Hypoxia-Inducible-Factor-1-α (HIF-1α) is stabilized and activates the expression of multiple genes contributing to the angiogenic 

process. HIF-1α induced proteins include Vascular Endothelial Growth Factor (VEGF) and Basic Fibroblast Growth Factor (bFGF), which promote vascular permeability and EC 

growth, respectively. Other secreted factors, such as PDGF, angiopoietin 1 and angiopoietin 2 facilitate chemotaxis, while ephrins guide newly formed blood vessels through 

maintenance of cell-cell separation. Other HIF-1α induced gene products include matrix metalloproteinases (MMPs) that breakdown the extracellular matrix to facilitate EC 

migration and release associated growth factors. Certain integrins such as αVβ3 found on the surface of angiogenic ECs help the sprouting ECs adhere to the provisional Extracellular 

Matrix (ECM), migrate and survive. Factors secreted into the microenvironment surrounding the tumor activate tumor-associated macrophages (TAMs), that subsequently 

produce angiogenic factors, such as VEGF and MMPs, further promoting angiogenesis. Pericytes function as support cells enveloping the basolateral surface of ECs and regulate 

vasoconstriction and dilation under normal physiologic conditions. During the process of tumor angiogenesis sprouting vessels lack pericytes, which are later recruited by 

ECs to provide structural support that indirectly promotes tumor survival. For example, PDGF secreted by ECs acts as a ligand for PDGF receptor located on the pericyte 

membrane, causing pericytes to produce and secrete VEGF that signals through the endothelial VEGF receptor. 


Folkman, J. (2007) Nat. Rev. Drug Disc. 6, 273–286. • Guo, C., Buranych, A., Sarkar, D., et al. (2013) Vasc. Cell. 5, 20. • Keith, B., Johnson, R.S., and Simon, M.C. 

(2012) Nat. Rev. Cancer 12, 9–22. • Klagsbrun, M. and Eichmann, A. (2005) Cytokine and Growth Factor Rev. 16, 535–548. • Raza, A., Franklin, M.J., and Dudek, A.Z. 

(2010) J. Hematol. 85, 593–598. • Sakurai, T. and Kudo, M. (2011) Oncology 81 Suppl. 1, 24–29. • Senger, D.R. and Davis, G.E. (2011) Cold Spring Harb. Perspect. Biol. 

3, a005090. • van Hinsbergh, V.W. and Koolwijk, P. (2008) Cardiovasc. Res. 78, 203–212. 

© 2008–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Diane Bielenberg, Harvard Medical School, Children’s Hospital, Boston, MA, for reviewing this diagram. 


07 


Syk is expressed 

in B cells and 

other cell lines. 

kDa 

140 

100 

80 

60 

50 

40 

30 

1 2 3 4 

Syk 

Syk (D3Z1E) XP ® Rabbit mAb #13198: 

WB analysis of extracts from various cell 

lines using #13198. 

Lanes 

1. SW620 

2. SR 

3. A20 

4. YB2/0 

Immunology and Inflammation 

B Cell and T Cell Receptor Signaling and Adaptive Immunity 

B and T lymphocytes mediate the humoral and cell-mediated immune responses, respectively, which 

make up the adaptive arm of the immune system. B cells mature in the bone marrow and differentiate 

into antibody-secreting plasma cells. In contrast, T cells are thymus-derived and, as effector cells, 

orchestrate cell-mediated immunity. 

The B cell receptor (BCR) is composed of a membrane-bound antibody (immunoglobulin or Ig) flanked 

by Igα/Igβ (CD79A/CD79B) heterodimers. When membrane Ig binds antigen, the CD79 heterodimer 

transduces signals through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) 

domains. The T cell receptor (TCR) consists of a membrane-bound αβ heterodimer (TCRαβ), four CD3 

chains (two CD3ε, one CD3γ, one CD3δ), and a ζ−chain homodimer. The TCRαβ dimer recognizes 

antigenic peptides, while the associated signaling chains transduce signals with their cytoplasmic ITAM 

domains. Thus, the lymphocyte antigen receptors use similar models of membrane-bound antigen 

receptors linked to signal-transducing accessory chains. 

Signaling through the BCR and TCR involves activation of a number of Src family tyrosine kinases (Blk, 

Fyn, and Lyn in B cells and Fyn and Lck in T cells), which are responsible for phosphorylation of the 

receptor-associated ITAM motifs. Phosphorylated ITAMs act as docking sites for Syk family tyrosine 

kinases (Syk in B cells and Zap-70 in T cells). Activated Syk kinases amplify signals through phosphorylation 

of downstream adaptor proteins, thereby initiating a cascade of intracellular signaling molecules. 

In addition to mediating cell activation, lymphocyte receptor signaling drives B and T cell development, 

differentiation, proliferation, and survival. 

Events 

Zap-70 is expressed in T cells. 

Zap-70 (D1C10E) XP ® Rabbit mAb (Alexa Fluor ® 488 Conjugate) #9473: Flow 

cytometric analysis of Ramos (B cells; blue) and Jurkat (T cells; green) cells using #9473. 

Jak/Stat Signaling 

The Jak/Stat signaling pathway is utilized by a large number of cytokines, growth factors, and hormones 

upon binding to their specific receptors. Receptor-mediated tyrosine phosphorylation of Jak family 

members triggers phosphorylation of Stat proteins, resulting in their nuclear translocation, binding 

to specific DNA elements, and subsequent activation of transcription. The remarkable range and 

specificity of responses regulated by the Stats is determined, in part, by the tissue-specific expression 

of different cytokine receptors, Jaks, and Stats, as well as by the combinatorial coupling of various 

Stat members to different receptors. Stat1 is activated in response to a large number of ligands and is 

essential for responsiveness to IFN-α and IFN-γ. Stat3 is constitutively activated in a number of human 

tumors and possesses both oncogenic potential and antiapoptotic activities. Stat4 has been most extensively 

investigated as a mediator of IL-12 responses. Stat5 is activated in response to a wide variety 

of ligands including IL-2, GM-CSF, growth hormone, and prolactin. 

Cytokine stimulation results in 

phosphorylation of Stat3 at Tyr705. 

Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb (Alexa Fluor ® 

488 Conjugate) #4323: Flow cytometric analysis of Jurkat cells, 

untreated (blue) or IFN-α treated (green), using #4323 compared to 

isotype control antibody (red). 

Growth factor 

stimulation results 


of Stat5 at Tyr694. 

A 

Events 

Phospho-Stat3 (Tyr705) 

(Alexa Fluor ® 488 Conjugate) 

B 

chapter 07: immunology and inflammation 

Phospho-Stat5 (Tyr694) (D47E7) 

XP ® Rabbit mAb #4322: Confocal 

IF analysis of A-431 cells, treated 

with Human Epidermal Growth Factor 

(hEGF) #8916 (A) or untreated (B), 

using #4322 (green) and Pan-Keratin 

(C11) Mouse mAb #4545 (red). 

Zap-70 

TLR Signaling and Innate Immunity 

The innate arm of the immune system consists of a host of immune cells and resistance mechanisms 

that act as the first line of defense against invading pathogens. The toll-like receptors (TLRs) are a family 

of evolutionarily conserved pattern recognition receptors (PRRs) that recognize the pathogen-associated 

molecular patterns (PAMPs) found in microbial pathogens. TLR1, 2, 4, 5, and 6 are expressed at the 

cell surface, while TLR3, 7, 8, and 9 have been shown to localize to intracellular vesicles. Activation 

of TLRs through ligand binding triggers a signaling cascade involving a variety of intracellular signaling 

adaptors including MyD88, IRAKs, and TRAF6. TLR signaling leads to the activation of the MAP kinase, 

NF-κB, and IRF signaling pathways, which mediate inflammation through the production of inflammatory 

cytokines, type I IFN, chemokines, and antimicrobial peptides. TLR signaling in innate immune cells, particularly 

dendritic cells, leads to their activation and subsequent induction of adaptive immune responses. 

TLR2 expression in mouse macrophages and dendritic cells 

Toll-like Receptor 2 (E1J2W) Rabbit mAb (Mouse Specific) #13744: WB analysis 

of extracts from Raw 264.7 cells, mouse bone marrow-derived macrophages (BMDM), 

and mouse bone marrow-derived dendritic cells (BMDC) using #13744. 

kDa 1 2 3 

200 


140 

100 

80 

60 

50 

TLR2 

Lanes 

1. Raw 264.7 

2. BMDM 

3. BMDC 

NF-κB Signaling 

Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and 

immune responses. There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), 

and NF-κB2 (p100/p52). Both p105 and p100 are co-translationally processed by the proteasome to 

produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that 

bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by 

IκB inhibitory proteins. NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting 

them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter 

the nucleus where it regulates gene expression. NIK and IKKα (IKK1) regulate the phosphorylation and 

processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus. 

NF-κB1 p105/p50 associates with promoters for 

IκBα and IL-8, but not with α satellite repeat element. 

NF-κB1 p105/p50 (D4P4D) Rabbit mAb #13586: Chromatin IPs were 

performed with cross-linked chromatin from 4 x 10 6 HeLa cells treated 

with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (30 ng/ml, 1 hr) 

and either 10 μl of #13586 or 2 μl of Normal Rabbit IgG #2729 using 

SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The 

enriched DNA was quantified by real-time PCR using SimpleChIP ® Human 

IκBα Promoter Primers #5552, human IL-8 promoter primers, and 

SimpleChIP ® Human α Satellite Repeat Primers #4486. The amount of 

immunoprecipitated DNA in each sample is represented as a percent of 

the total input chromatin. 

NF-κB1 p105/p50 (D4P4D) 


Normal Rabbit 

IgG #2729 


4.0 

3.5 

3.0 

2.5 

2.0 

1.5 

1.0 

0.5 

0 

IκB-α 

IL-8 

α Satellite 

TNF-α treatment 

results in translocation 

of NF-κB p65 (RelA) to 

the nucleus. 

NF-κB p65 (D14E12) XP ® Rabbit 


HT-1080 cells, untreated (top) or treated 

with hTNF-α #8902 (20 ng/ml, 20 min) 

(bottom), using #8242 (green). Actin 

filaments were labeled with DY-554 



www.cellsignal.com/cstimmunology 173



PD-L1 is expressed 

in lung carcinoma. 

PD-L1 (E1L3N ® ) XP ® Rabbit mAb 



These protein targets represent 

key nodes within immunology and 

inflammation signaling pathways and 

are commonly studied in immunology 

and inflammation research. Primary 











Immune Checkpoints 

Activation of T lymphocytes by antigen-presenting cells (APCs) requires engagement of the T cell 

receptor with MHC class I or II molecules and co-stimulatory signals generated from CD28 (on T cells) 

binding to CD80 or CD86 (on APCs). However, under certain circumstances, such as maintaining 

self-tolerance or preventing collateral tissue damage, T cell engagement is coupled with inhibitory 

signals that repress T cell activation and response, known as immune checkpoints. Immune checkpoint 

proteins such as PD-1 and CTLA-4, which are commonly upregulated in infitrating T cells, bind their 

corresponding ligands, PD-L1 and CD80/86 respectively, which are upregulated in cancer cells as a 

means to evade immune detection and downregulate T cell response. Activating antitumor immunity 

through the blockade of immune checkpoint proteins has become a promising therapeutic strategy for 

the treatment of cancer. 


Borroto, A., Abia, D., and Alarcón, B. (2014) Immunol. Lett. 161, 113−117. • Burger, J.A. and Chiorazzi, N. (2013) Trends Immunol. 

34, 592−601. • Dorritie, K.A., Redner, R.L., and Johnson, D.E. (2014) Adv. Biol. Regul. 56, 30–44. • Fu, G., Rybakin, V., 

Brzostek, J., et al. (2014) Trends Immunol. 35, 311−318. • Gasparini, C., Celeghini, C., Monasta, L., et al. (2014) Cell Mol. Life 

Sci. 71, 2083−2102. • Gerondakis, S., Fulford, T.S., Messina, N.L., et al. (2014) Nat. Immunol. 15, 15−25. • Harwood, N.E. and 

Batista, F.D. (2010) Annu. Rev. Immunol. 28, 185–210. • Kawakami, Y., Yaguchi, T., Park, J.H., et al. (2013) Front. Oncol. 3, 136. 

• Maddaly, R., Pai, G., Balaji, S., et al. (2010) FEBS Lett. 584, 4883–4894. • Mifsud, E.J., Tan, A.C., and Jackson, D.C. (2014) 

Front. Immunol. 5, 79. • Pardoll, D.M. (2012) Nat. Rev. Cancer 12, 252–264. • Reuven, E.M., Fink, A., and Shai, Y. (2014) 

Biochim. Biophys. Acta. 1838, 1586−1593. • Vanneman, M. and Dranoff, G. (2012) Nat. Rev. Cancer 12, 237−251. 

Commonly Studied Immunology and Inflammation Targets 


A20/TNFAIP3 • • 

ABIN-1 • • 

ADAP • 

AID • 

AIM2 • • 

Aiolos • 

AML1 • • 

Phospho-AML1 

(Ser249) 

• 

β2-microglobulin • • • 

BACH2 • 

BAFF • 

Basigin/EMMPRIN • • 

BATF • 

BCL6 • • 

Bcl10 • 

Blimp-1/PRDI-BF1 • 

Blk • 

BLNK • 

Phospho-BLNK 

(Tyr96) • 

Btk • • 

• 

Phospho-Btk 

(Ser180) 

Phospho-Btk 

(Tyr223) 

CARD9 

CARD11 

Phospho-CARD11 

(Ser652) 

CBFβ 

CCR2 

CD3ε 

CD4 

CD8 

• 

• 

• • 

• 

• 

• 

• 

• 

• 


CD9 • 

CD10 • 

CD13 • 

CD19 • 

Phospho-CD19 

(Tyr531) • 

CD31 (PECAM-1) • 

CD34 • 

CD44 • • • • 

CD45 • 

CD46 • 

CD79A • • 

Phospho-CD79A 

(Tyr182) • 

CD82 • • 

CIITA • 

CISH • 

CrkL • 

• 

Phospho-CrkL 

(Tyr207) 

Cytokine Receptor 

Common β-Chain 

Cox1 

• 

• • 

Cox2 • • • • 

Cyclophilin A • 

DAP12 • 

DC-SIGN 

Dectin-1 

E2A 

ERC1 

ERC1α 

ETO 

Evi-1 

Fgr 

FoxP3 

• • 

• 

• • 

• 

• 

• 

• • 

• 

• 


Fyn • • 

Galectin-1/LGALS1 • • 

Galectin-3/LGALS3 • 

GIMAP5 • 

GP130 • 

GRK6 • 

Helios • 

HPK1 • 

HS1 • • 

Phospho-HS1 

(Tyr397) • • • 

IFI16 • 

IFIT1 • 

IFITM1 • 

IFITM2 • 

IFN-α • 

IFN-γ • • 

IGBP1 • 

Ikaros • • 

IκBα • • • • 

Phospho-IκBα 

(Ser32) • • • 

Phospho-IκBα 

(Ser32/Ser36) 

IκBα (Aminoterminal 

Antigen) • • 

IκBα (Carboxyterminal 

Antigen) 

IκBβ 

Phospho-IκBε 

(Ser18/22) 

IκBζ 

IKKα 

Phospho-IKKα 

(Ser176)/IKKβ 

(Ser177) 

• 

• • 

• 

• 

• • • • 

• 


• 


Phospho-IKKα/β 

(Ser176/180) • • 

IKKβ • • • 

• 

Phospho-IKKβ 

(Ser177/181) 

IKKγ 

Phospho-IKKγ 

(Ser376) 

IKKε 

Phospho-IKKε 

(Ser172) 

IL-1β 

IL-1RA 

IL-2 

IL-2Rα 

IL-2Rβ 

Mouse IL-3 

Neutralizing 

Human IL-4 

Neutralizing 

IL-4 

IL-6 

IL10 

IL-17A 

Human IL-17A 

Neutralizing 

IL17R 

IDO 

• • 

• 

• • 

• 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• 

• 

• 

• • 

• 

IRAK1 • • • • 

• 

Phospho-IRAK1 

(Thr209) 

Phospho-IRAK1 

(Thr387) 

IRAK2 

IRAK4 

Phospho-IRAK4 

• 

• 

• 

(Thr345/Ser346) • • 

IRAK-M • 

IRF-1 • • 

IRF-2 • 

IRF-3 • • 

Phospho-IRF-3 

(Ser396) • 

IRF-4 • • 

IRF-5 • • 

IRF-6 • 

IRF-7 • • • 

• 

Phospho-IRF-7 

(Ser471/472) 

Phospho-IRF-7 

(Ser477) 

IRF-8 

Itk 

Jak1 

Phospho-Jak1 

(Tyr1022/1023) 

• 

• 

• 

• • 

• 

Jak2 • • • 

• 

Phospho-Jak2 

(Tyr221) 

Phospho-Jak2 

(Tyr1007) 

Phospho-Jak2 

(Tyr1007/1008) 

• 

• • 


Phospho-Jak2 

(Tyr1008) • • 

Jak3 • • 

Phospho-Jak3 

(Tyr980/Tyr981) • 

Langerin 

LAT 

• 

• 

• 

Phospho-LAT 

(Tyr171) 

Phospho-LAT 

(Tyr191) 

Lck 

Phospho-Lck 

(Tyr505) 

LGP2 

LITAF 

5-Lipoxygenase 

Phospho-5-Lipoxygenase 

(Ser271) 

Phospho-5-Lipoxygenase 

(Ser663) 

LRF/Pokemon 

Lsp1 

Lyn 

Phospho-Lyn 

(Tyr507) 

MALT1 

Mannose Receptor 

MAVS 

MCP-1 mouse 

MDA-5 

MEIS1/2 

• • 

• • 

• • 

• 

• 

• 

• 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• 

• 

Miz-1 • • 

MNDA • • 

MyD88 • • 

Myeloperoxidase • 

NALP1 • 

NDP52 • • 

NFAT1 • • 

NFAT2 • 

NFAT3 • 

NFAT4 • 

NF-κB p65 • • • • 

• 

Phospho-NF-κB 

p65 (Ser468) 


p65 (Ser536) • • • • 

Acetyl-NF-κB p65 

(Lys310) 

Methyl-NF-κB p65 

(Lys310) 

NF-κB p105 


p105 (Ser932) 

NF-κB p105/p50 

• • 

NF-κB2 p100/p52 • • 

Phospho-NF-κB2 

p100/p52 

(Ser866/Ser870) 

NIK 

NLRC4 

NLRP3 

NLRX1 

Nod1 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• 


NOS (pan) • 

iNOS • • • 

NTAL/LAB • • 

Phospho-p40phox 

(Thr154) • 

p47phox • • 

p67phox • 

Pbx1 • 

PD-L1 • 

PIAS1 • 

PIAS3 • • • 

PIAS4 • 

Pim-1 • • 

Pim-2 • 

Pim-3 • 

Pirin • 

Prolactin Receptor • 

PTPN22 • 

PU.1 • • • 

RAG1 • 

RAGE • 

RAGE 1 • • 

RANK • 

RANK Ligand • 

RANTES • 

c-Rel • • • 

RelB • • 

Phospho-RelB 

(Ser552) • • • 

Rig-I • • 

RIP • • 

RIP2 • • 

Phospho-RIP2 

(Ser176) • • 

RIP3 • • 

RIP4 • 

SAMHD1 • 

SARM1 • 

SDF1 • • 

SH2D1A • • 

SHIP1 • • 

Phospho-SHIP1 

(Tyr1020) • 

SHIP2 • • 

• 

Phospho-SHIP2 

(Tyr986/Tyr987) 

Phospho-SHIP2 

(Tyr1135) 

SHP-1 

Phospho-SHP-1 

(Tyr564) 

SINTBAD 

SLP76 

Phospho-SLP76 

(Ser376) 

SOCS1 

SOCS2 

SOCS3 

• 

• 

• 

• 

• 

• 

• 

• 

• 

Stat1 • • • 

Phospho-Stat1 

(Tyr701) • • • 

285 


CST antibodies for Phospho-Stat3 

(Tyr705) have been cited over 285 times 




Mauer, J. et al. (2014) Signaling by 

IL-6 promotes alternative activation 

of macrophages to limit endotoxemia 

and obesity-associated resistance to 

insulin. Nat. Immunol. 15, 423−430. 

Kothari, P. et al. (2014) IL- 

6-mediated induction of matrix 

metalloproteinase-9 is modulated 

by JAK-dependent IL-10 expression 

in macrophages. J. Immunol. 192, 

349−357. 

Zhou, C. et al. (2014) PTTG acts as 

a STAT3 target gene for colorectal 

cancer cell growth and motility. 

Oncogene 33, 851−861. 

Kryczek, I. et al. (2014) IL-22(+) 

CD4(+) T cells promote colorectal 

cancer stemness via STAT3 transcription 

factor activation and induction 

of the methyltransferase DOT1L. 

Immunity 40, 772−784. 

Mayeur, C. et al. (2014) The type I 

BMP receptor Alk3 is required for the 

induction of hepatic hepcidin gene 

expression by interleukin-6. Blood 

123, 2261−2268. 

Becker, T.M. et al. (2014) Mutant B- 

RAF-Mcl-1 survival signaling depends 

on the STAT3 transcription factor. 

Oncogene 33, 1158−1166. 

Xiang, M. et al. (2014) STAT3 induction 

of miR-146b forms a feedback 

loop to inhibit the NF-kappaB to IL-6 

signaling axis and STAT3-driven cancer 

phenotypes. Sci. Signal. 7, ra11. 

Wang, Y. et al. (2014) GdX/UBL4A 

specifically stabilizes the TC45/ 

STAT3 association and promotes 

dephosphorylation of STAT3 to 

repress tumorigenesis. Mol. Cell 53, 

752−765. 

Schmidt, J.W. et al. (2014) Stat5 

regulates the phosphatidylinositol 

3-kinase/Akt1 pathway during 

mammary gland development and 

tumorigenesis. Mol. Cell Biol. 34, 

1363−1377. 

Nagashima, H. et al. (2014) The 

adaptor TRAF5 limits the differentiation 

of inflammatory CD4(+) T cells 

by antagonizing signaling via the 

receptor for IL-6. Nat. Immunol. 15, 

449−456. 

www.cellsignal.com/cstimmunology 175



Jak and Cytokine 

Receptor Mutants 

This table lists Jak and cytokine receptor 

mutations found in various cancers, 

along with corresponding publications. 


Phospho-Stat1 

(Ser727) • • 

Stat2 • 

Phospho-Stat2 

(Tyr690) • 

Stat3 

• • • • • 

Phospho-Stat3 

(Tyr705) • • • • 

Phospho-Stat3 

(Ser727) 

Acetyl-Stat3 

(Lys685) 

Stat3α 

Stat4 

• • • 

• 

• • 

• 

Phospho-Stat4 

(Tyr693) • • • 

Stat5 • • • • 

Phospho-Stat5 

(Tyr694) • • • • 

Stat5a 

Stat6 

• 

• • • • 

Phospho-Stat6 

(Tyr641) • • • • 

STING 

Syk 

• 

• • 

• 

Phospho-Syk 

(panTyr) 


Phospho-Syk 

(Tyr323) • 

Phospho-Syk 

(Tyr525/526) • • • • 

TAL1 • 

TAP1 • 

TAP2 • 

T-bet/TBX21 

(V365) • • 

TBK1/NAK • • 

Phospho-TBK1/ 

NAK (Ser172) • • 

Tec • 

THEMIS • 

ThPOK • 

TIRAP • 

Toll-like Receptor 1 • 

Toll-like Receptor 2 • • 







• 

• 

Human TNF-α 

Neutralizing 


Mouse TNF-α 

Neutralizing • 

TNF-α • • • • 

TNF-R1 • • 

Tollip • 

Phospho-TPOR 

(Tyr626) • 

TREX1 • 

TRIF • 

TWEAK • 

TWEAK Receptor/ 

Fn14 • 

Tyk2 • • • 

Phospho-Tyk2 

(Tyr1054/1055) • 

VCAM1 • 

Yes • 

ZAP70 • • • 

• 

Phospho-ZAP70 

(Tyr319) 

Phospho-Zap-70 

(Tyr319)/Syk 

(Tyr352) 

Phospho-ZAP70 

(Tyr493) 

• • • 

• 

Jak Mutants Cytokine Receptor Disease References 

Jak2 V617F EpoR, TpoR (MPL), G-CSFR Myeloproliferative neoplasms 1–5 

(MNPs), PV, ET, PMF 

Jak2 K539L, exon 12 mutants EpoR MNP: PV 6 

Jak2 T875N Undetermined AML (AMKL) 7 

Jak3 A572V Undetermined AML (AMKL) (cell lines) 8 

Jak1 V658F, Jak1 A634D, R879H, R724S IL2R, IL9R, other undetermined T-ALL 9,10 

Jak1 R683G/S Jak2 DIREED TLSPR Pediatric and Down syndrome ALL 11–15 

Jak2 V617I, Jak2 R564Q, Jak2 S755R/ R938Q TpoR (MPL) Hereditary thrombocytosis 16–18 

Receptor Mutants Cytokine Receptor Disease References 

TpoR W515L/K/A Jak2 MPNs: ET, PMF 19–21 

TpoR S505N 22 

TpoR S487A 23 

TLSPR F232S / TLSPR translocations Jak2 R683 mutants Pediatric and Down syndrome ALL 13, 24–26 

References: (1) James, C. et al. (2005) Nature 434, 1144–1148. • (2) Baxter, E.J. et al. (2005) Lancet 365, 1054–1061. • 

(3) Kralovics, R. et al. (2005) N. Engl. J. Med. 352, 1779–1790. • (4) Levine, R.L. et al. (2005) Cancer Cell 7, 387–397. • (5) 

Vainchenker, W. et al. (2008) Semin. Cell Dev. Biol. 19, 385–393. • (6) Scott, L.M. et al. (2007) N. Engl. J. Med. 356, 459–468. 

• (7) Mercher, T. et al. (2006) Blood 108, 2770–2779. • (8) Walters, D.K. et al. (2006) Cancer Cell 10, 65–75. • (9) Flex, E. et 

al. (2008) J. Exp. Med. 205, 751–758. • (10) Jeong, E.G. et al. (2008) Clin. Cancer Res. 14, 3716–3721. • (11) Malinge, S. 

et al. (2007) Blood 109, 2202–2204. • (12) Mullighan, C.G. et al. (2009) Proc. Natl. Acad. Sci. USA 106, 9414–9418. • (13) 

Mullighan, C.G. et al. (2009) Nat. Genet. 41, 1243–1246. • (14) Bercovich, D., et al. (2008) Lancet 372, 1484–1492. • (15) 

Kearney, L. et al. (2009) Blood 113, 646–648. • (16) Mead, A.J. et al. (2013) Blood 121, 4156–4165. • (17) Etheridge, S.L. 

et al. (2014) Blood 123, 1059–1068. • (18) Marty, C. et al. (2014) Blood 123, 1372–1383. • (19) Pikman, Y. et al. (2006) 

PLoS Med. 3, e270. • (20) Pardanani, A.D. et al. (2006) Blood 108, 3472–3476. • (21) Pecquet, C. et al. (2010) Blood 115, 

1037–1048. • (22) Ding, J. et al. (2004) Blood 103, 4198–4200. • (23) Malinge, S. et al. (2008) Blood 112, 4220–4226. 

• (24) Russell, L.J. et al. (2009) Blood 114, 2688–2698. • (25) Chapiro, E. et al. (2010) Leukemia 24, 642–645. • (26) 

Hertzberg, L. et al. (2010) Blood 115, 1006–1017. 

Ligand Receptor Jak-Kinase Other Tyrosine Kinases Stat Family Members 

IL-6 IL-6Rα+gp130 Jak1,2, Tyk2 Hck Stat1, Stat3 

IL-11 IL-11R+gp130 Jak1,2, Tyk2 Src, Yes Stat3 

CNTF, CT-1, LIF, OSM CNTFR+gp130, CT-1R+gp130, 

LIFR+gp130, OSMR+gp130 

Jak1,2, Tyk2 Src family Predominant: Stat3 

Secondary: Stat1,5 

G-CSF G-CSFR Jak2, Tyk2 Lyn Stat3 

IL-12 (p40+p35) IL-12Rβ1+IL-12Rβ2 Jak2, Tyk2 Lck Stat4 

Leptin LeptinR Jak2 Not determined Stat3,5,6 

IL-3 IL-3Rα+βc Jak2 Fyn, Hck, Lyn Stat3,5,6 

IL-5 IL-5R+βc Jak2 Btk Stat3,5,6 

GM-CSF GM-CSFR+βc Jak2 Hck, Lyn Stat3,5 

Angiotensin GPCR Jak2, Tyk2 Stat1,2,3 

Serotonin GPCR Jak2 Stat3 

α-Thrombin GPCR Jak2 Stat1,3 

Chemokines CXCR4 Jak2,3 

IL-2 IL-2Rα+IL-2Rb+γc Jak1,2,3 Fyn, Hck, Lck, Syk, Tec Stat3,5 

IL-4 IL-4Rα+γcR or IL-4Rα+IL-13Rα1 Jak1,3 Lck, Tec Stat6 

IL-7 IL-7R+γc Jak1,3 Lyn Stat3,5 

IL-9 IL-9R+γc Jak1,3 Not determined Stat1,3,5 

IL-13 IL-13Rα1+ IL-4Rα Jak1,2, Tyk2 Ctk Stat6 

IL-15 IL-15Rα+IL-2Rβ+γc Jak1,3 Lck Stat3,5 

IL-19 IL-20Rα+IL-20Rβ Jak1, Stat3 

IL-20 IL-20Rα+IL-20Rβ, IL-22R+IL-20Rβ Jak1, Stat3 

IL-21 IL-21R+γc Jak1,3 Stat1,3,5 

IL-22 IL-22R+IL-10Rβ Jak1, Tyk2 Stat1,3,5 

IL-23 (p40+p19) IL-12Rβ1+IL-23R Jak2 Tyk2 Stat4 

IL-24 same as IL-20 Jak1, Stat3 

IL-26 IL-20Rα+IL-10Rβ Jak1, Tyk2 Stat3 

IL-27 (EBI3+p28) gp130+WSX1 Jak1,2, Tyk2 Stat1,2,3,4,5 

IL-28A, IL-28B, IL-29 IL-28R+IL-10Rβ Jak1, Tyk2 Stat1,2,3,4,5 

IL-31 IL-31Rα+OSMR Jak1,2, Tyk2 Stat1,3,5 

IL-35 (p35+EBI3) gp130+WSX1 Jak1,2, Tyk2 Stat1,3,5 

GH GHR Jak2 Src family Stat3,5 (mainly Stat5a) 

Tpo TpoR (c-Mpl) Jak2, Tyk2 Lyn Stat1,3,5 

Epo, Pro EpoR, ProlactinR Jak2 Src Family Stat5 (mainly Stat5a) 

Interferon (IFNα/β) IFNAR1+IFNAR2 Jak1, Tyk2 Lck Predominant: Stat1,2 

Secondary: Stat3,4,5 

IFN-γ IFN-gR1+IFN-γR2 Jak1, Jak2 Hck, Lyn Stat1 

IL-10 IL-10Rα+ IL-10Rβ Jak1, Tyk2 Not determined Stat1,3,5 

TLSP TLSPR and IL-7R Jak1, possibly 

Jak2 

Not determined Stat3,5 

EGF EGFR Jak1 EGFR, Src Stat1,3,5 

PDGF PDGFR Jak1,2 PDGFR, Src Stat1,3,5 


We would like to thank Prof. Stefan Constantinescu, Ludwig Institute for Cancer Research, Brussels, Belgium for contributing to this table. 

Jak/Stat 

Utilization 

This table lists the combinatorial use 

of tyrosine kinases and Stat proteins 

in cytokine/growth factor signaling. 

Protein Kinases: Introduction 

Please visit our website to learn more about Protein Kinases. www.cellsignal.com/cstkinases 



177

CRAC Channel 

Orai1 



B Cell Receptor Signaling 

BCR 


p38 

JNK 

BCR 

Ag 

Gab 

Shc 

GRB2 

Lyn CD22 SHP-1 

SHP-2 

p85 p110 

PI3K PIP 3 

FcγRIIB1 

Lipid 

Lyn 

Raft Ezrin CD19 

Aggregation 

Syk 

SHIP 

SHP-1 

PTEN 

CD45 

PIR-B 

RhoA 

Dok-3 

clathrin 

SHP-2 

PI(4,5)P 2 

Btk 

Ca 2+ 

Bam32 Cbp/PAG 

Cbl 

IP3R 

Csk 

Bam32 

STIM1 

PLCγ2 

Intracellular 

IP Ca 2+ 3 

Store 

Akt 

Ca 

LAB 

2+ 

CIN85 

GRB2 

DAG 

Vav 

SOS 

Rac/ 

PKC 

cdc42 CD19 HS1 

Ras 

PIP 3 

GRP 

Pyk2 

DAG Ras 

CARMA1 

Ras TAK1 

Rac 

GAP 

Bcl10 

RapL 

MALT1 

c-Raf 

Rap 

CaM GSK-3 mTOR 

Riam 

Dok-1 

MEKKs 

FcγRIIB1 

IKK 

DAG 

Cofilin 

CD19 MEK1/2 

Calcineurin 

MKK3/4/6 MKK4/7 

p70 

IκB 

S6K 

PKC Ca 2+ Erk1/2 

NF-κB 

CD40 

IκB 

p38 

NFAT Protein 

JNK1/2 

Proteasomal 

Synthesis 

Degradation 

Cytoskeletal 

Rearrangements and 

Integrin Activation 

Cytoplasm 

Nucleus 

Transmembrane Protein 

mIg 

α/β α/β 

BLNK 

GRB2 

LAB 

Erk1/2 

mIg 

BCAP 

NF-κB 

CD19 

CaMK 

Ca 2+ 

Glucose 

Uptake 

Glycolysis 

ATP 

Generation 

Akt 

T Cell Receptor Signaling 

Ca 2+ Ca 2+ Ca 2+ Clustering 

CRAC 

Channel 

Calcineurin 

CaM 

NFAT 

CaMKIV 

CREB 

NFAT 

LFA-1 

Actinin Talin 

Calpain 

F-Actin 

IP 3 

IP3R 

Intracellular 

Ca 2+ Store 

SOS 

PIP 2 

DAG 

RasGRP 

Ras 

Raf 

MEK1/2 

Erk1/2 

GRB2 

LAT 

GADS 

PLCγ1 

NCK 

Itk 

SLP-76 

WASP 

Rac/cdc42 

MEKK1 

MKK4/7 

JNK 

ADAP 

Vav 

TAK1 

HPK1 

GADD45α 

ζ ζ 

Zap-70 

Dlgh1 

p38 MAPK 

α β 

c-Cbl 

DAG 

TAK1 

MKK7 

δ ε ε γ 

JNK2 

TCR/CD3 

Complex 

PDK1 

PKCθ 

MALT1Bcl10 

Carma1 

IKKβ 

IKKγ 

CD4 

Lck 

IKKα 

Rel 

IκB 

NF-κB 

IκB 

CD45 

GβL 

Proteasomal 

Degradation 

CD28 

PI3K 

PIP 3 

Akt 

mTORC1 

Raptor 

mTOR 

DEPTOR 

Cell Proliferation 

Survival 

MEF2C 

CREB 

ATF-2 Jun Bcl-6 Egr-1 Elk-1 Bcl-xL Bfl-1 Oct-2 Ets-1 NFAT 

Transcription 

FoxO 

Transcription 

Growth Arrest, 

Apoptosis 

Cytoplasm 

Nucleus 

IL-2 

Gene 

NFAT Fos 

Jun NF-κB Rel 

Nuclear 

Membrane 

The B cell antigen receptor (BCR) is composed of membrane immunoglobulin (mIg) molecules and associated Igα/Igβ (CD79a/CD79b) heterodimers (α/β). The mIg subunits 

bind antigen, resulting in receptor aggregation, while the α/β subunits transduce signals to the cell interior. BCR aggregation rapidly activates the Src family kinases Lyn, 

Blk, and Fyn as well as the Syk and Btk tyrosine kinases. This initiates the formation of a ‘signalosome’ composed of the BCR, the aforementioned tyrosine kinases, adaptor 

proteins such as CD19 and BLNK, and signaling enzymes such as PLCγ2, PI3K, and Vav. Signals emanating from the signalosome activate multiple signaling cascades that 

involve kinases, GTPases, and transcription factors. This results in changes in cell metabolism, gene expression, and cytoskeletal organization. The complexity of BCR signaling 

permits many distinct outcomes, including survival, tolerance (anergy) or apoptosis, proliferation, and differentiation into antibody-producing cells or memory B cells. The 

outcome of the response is determined by the maturation state of the cell, the nature of the antigen, the magnitude and duration of BCR signaling, and signals from other 

receptors such as CD40, the IL-21 receptor, and BAFF-R. Many other transmembrane proteins, some of which are receptors, modulate specific elements of BCR signaling. 

A few of these, including CD45, CD19, CD22, PIR-B, and FcγRIIB1 (CD32), are indicated here in yellow. The magnitude and duration of BCR signaling are limited by negative 

feedback loops including those involving the Lyn/CD22/SHP-1 pathway, the Cbp/Csk pathway, SHIP, Cbl, Dok-1, Dok-3, FcγRIIB1, PIR-B, and internalization of the BCR. In 

vivo, B cells are often activated by antigen-presenting cells that capture antigens and display them on their cell surface. Activation of B cells by such membrane-associated 

antigens requires BCR-induced cytoskeletal reorganization. Please refer to the diagrams for the PI3K/Akt signaling pathway, the NF-κB signaling pathway, and the regulation 

of actin dynamics for more details about these pathways. 


Dal Porto, J.M., Gauld, S.B., Merrell, K.T., et al. (2004) Mol. Immunol. 41, 599–613. • Goodnow, C.C., Vinuesa, C.G., Randall, K.L., et al. (2010) Nat. Immunol. 11, 

681–688. • Harwood, N.E. and Batista, F.D. (2010) Annu. Rev. Immunol. 28, 185–210. • Harwood, N.E. and Batista, F.D. (2008) Immunity 28, 609–619. • Kurosaki, T., 

Shinohara, H., and Baba, Y. (2010) Annu. Rev. Immunol. 28, 21–55. • Szydłowski, M., Jabłońska, E., and Juszczyński, P. (2014) Int. Rev. Immunol. 33, 146–157. 

Tumor cells employ multiple defense strategies to evade detection by the immune system. One common strategy, upregulation of immune checkpoint proteins and ligands, takes 

advantage of a natural immune mechanism for self-tolerance and prevention of collateral tissue damage. Immune checkpoint proteins, such as PD-1, CTLA-4, and many others, 

are located on T cells and engage with their corresponding ligand on tumor cells or dendritic cells, sending inhibitory signals that repress T cell activation or response. One of 

the first discovered checkpoint proteins, CTLA-4, plays a role at the stage of T cell priming by binding to the CD28 ligands CD80 or CD86 to prevent co-stimulatory signals 

necessary for T cell activation. In contrast, the PD-1/PD-L1 checkpoint acts later in the process, inhibiting anti-tumor immune responses by effector T cells such as CD4 + T 

helper 1 (Th1) cells and CD8 + cytotoxic T lymphocytes (CTLs), leading to decreases in IFNγ production and cytolytic activity. Upregulation of PD-L1 expression on the tumor cell 

surface is mediated by IFNγR signaling to Stat1, as well as oncogenic signaling through several receptor tyrosine kinases (EGFR, ALK, ROS, HER2, and others) to activate the 

MAPK, Akt, and Stat3 pathways. 

Cells in the tumor microenvironment can also influence tumor progression. FoxP3 + /CD4 + T regulatory cells (T Regs ) and myeloid-derived suppressor cells (MSCs) secrete immunosuppressive 

cytokines IL-10 and TGF-β to inhibit the activity of Th1 cells and CTLs. Natural killer (NK) cells release cytotoxic granules against the tumor cell and secrete 

IFNγ, which stimulates surrounding pro-inflammatory M1 macrophages. Pro-tumorigenic M2 macrophages suppress anti-tumor immune responses via production of IL-10 

and TGF-β and promote metastasis through release of MMPs. MMPs and TGF-β are also released by surrounding mast cells. 


Burbach, B.J., Medeiros, R.B., Mueller, K.L., and Shimizu, Y. (2007) Immunol. Rev. 218, 65–81. • Chen, L. and Flies, D.B. (2013) Nat. Rev. Immunol. 13, 227–242. • 

Cheng, J., Montecalvo, A., and Kane, L.P. (2011) Immunol. Res. 50, 113–117. • Cronin, S.J. and Penninger, J.M. (2007) Immunol. Rev. 220, 151–168. • Fracchia, K.M., 

Pai, C.Y., and Walsh, C.M. (2013) Front Immunol. 4, 324. • Marsland, B.J. and Kopf, M. (2008) Trends Immunol. 29, 179–185. • Thome, M. (2008) Nat. Rev. Immunol. 8, 

495–500. 

© 2002–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Michael R. Gold, University of British Columbia, Vancouver, British Columbia, Canada for reviewing this diagram. 


© 2004–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Sankar Ghosh, Columbia University, New York, NY for reviewing this diagram. 




Toll-like Receptor Signaling 

UNC93B1 

TLR9 

TLR9 

MyD88 

TLR7 

LPS 

dsRNA or 

5'-triphosphate RNA 

CpG 

ATP 

MAVS 

Anti-viral 

Compounds, 

ssRNA 

MyD88 

CD14 

TLR8 

MD-2 

TLR4 

TLR4 

TRAM TRIF TIRAP 

RIG-I 

MyD88 

IRF-7 

IRF-7 

SARM1 

Endosome 

UNC93B1 UNC93B1 

dsRNA 

TLR3 

TLR3 

MyD88 

TRAF3 

TRIF 

SARM1 

IRF-3 

IRF-3 

Uropathogenic 

Bacteria, 

Profilin 

ST2L 

TLR11 

TLR11 

MyD88 

TRIAD3A 

SOCS1 

IKKε 

TBK1 

TOLLIP 

RIP1 

Proteasomal 

Degradation 

Flagellin 

TLR5 

TLR5 

MyD88 

IRAK-4 

IRAK-1 

TRAF6 

Ubc13 

UEV1A 

IKKγ/ 

NEMO 

IKKβ IKKα 

MyD88 

IRAK-M 

IκBα ub 

p65/RelA 

NF-κB 

TIRAP 

IRAK-2 

A20 

ECSIT 

Triacyl 

Lipopeptide 

TLR2 

TLR1 

TAK1 

TAB1/2 

MKK 4/7 

JNK 

MyD88 

FADD 

MEKK-1 

TIRAP 

MKK 3/6 

p38 MAPK 

Diacyl 

Lipopeptide 

TLR2 

TLR6 

Casp-8 

Apoptosis 

Jak/Stat Signaling: IL-6 Receptor Family 

p120 

ras-GAP 

E-Ras 

PI3K 

Akt 

Tumor-like 

Properties 

of ES Cells 

Cytoplasm 

Nucleus 

Erk 

Raf 

MEK 

Erk 

C/EBPβ 

NF-κB 

AP-1 

etc. 

Erk 

Ras 

Erk 

Shc 

GRB2 

TF 

TF 

Accessory 

TF Motif 

PI3K 

Akt 

mTOR 

SHP-2 

SOCS3 

Jak 

gp130 

Stat3 

Stat3 

Stat3 

Jak 

Stat3 

ISRE/GAS 

Mcl-1 

IL-6 

Apoptosis 

PIAS 

SUMO 

PTP 

SHP-1 

Stat 

SOCS 

Renewal 

of ES Cells 

Crosstalk 

Tumor Cells 

EGF 

EGFR 

Feedback 

Inhibition 

Stat 

Src 

CIS, SOCS, Mcl-1, APPs, TIMP-1, 

Pim-1, c-Myc, cytokines, TFs, etc. 

Cytoplasm 

Nucleus 

Inflammation, Immune Regulation, Survival, Proliferation 

Transcription 

Factors 

Toll-like receptors (TLRs) recognize distinct pathogen-associated molecular patterns and play a critical role in innate immune responses. They participate in the first line of 

defense against invading pathogens and play a significant role in inflammation, immune cell regulation, survival, and proliferation. To date, 11 members of the TLR family 

have been identified, of which TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 are located on the cell surface and TLR3, TLR7, TLR8, and TLR9 are localized to the endosomal/ 

lysosomal compartment. The activation of the TLR signaling pathway originates from the cytoplasmic Toll/IL-1 receptor (TIR) domain that associates with a TIR domain-containing 

adaptor, MyD88. Upon stimulation with ligands, MyD88 recruits IL-1 receptor-associated kinase-4 (IRAK-4) to TLRs through interaction of the death domains of both 

molecules. IRAK-1 is activated by phosphorylation and associates with TRAF6, thereby activating the IKK complex and leading to activation of MAP kinases (JNK, p38 MAPK) 

and NF-κB. Tollip and IRAK-M interact with IRAK-1 and negatively regulate the TLR-mediated signaling pathways. Additional modes of regulation for these pathways include 

TRIF-dependent induction of TRAF6 signaling by RIP1 and negative regulation of TIRAP-mediated downstream signaling by ST2L, TRIAD3A, and SOCS1. Activation of MyD88- 

independent pathways occurs via TRIF and TRAF3, leading to recruitment of IKKε/TBK1, phosphorylation of IRF3, and expression of interferon-β. TIR domain containing 

adaptors such as TIRAP, TRIF, and TRAM regulate TLR-mediated signaling pathways by providing specificity for individual TLR signaling cascades. TRAF3 plays a critical role in 

the regulation of both MyD88-dependent and TRIF-dependent signaling via TRAF3 degradation, which activates MyD88-dependent signaling and suppresses TRIF-dependent 

signaling (and vice versa). 


Barton, G.M. and Kagan, J.C. (2009) Nat. Rev. Immunol. 9, 535–542. • Blasius, A.L. and Beutler, B. (2010) Immunity 32, 305–315. • Kawai, T., and Akira, S. (2010) Nat. 

Immunol. 11, 373–384. • Lester, S.N. and Li, K. (2014) J. Mol. Biol. 426, 1246–1264. • Li, X., Jiang, S., and Tapping, R.I. (2010) Cytokine 49, 1–9. • McGettrick, A.F. 

and O’Neill, L.A. (2010) Curr. Opin. Immunol. 22, 20–27. • Miggin, S.M. and O’Neill, L.A. (2006) J. Leukoc. Biol. 80, 220–226. • Pasare, C. and Medzhitov, R. (2005) 

Adv. Exp. Med. Biol. 560, 11–18. • Reuven, E.M., Fink, A., and Shai, Y. (2014) Biochim. Biophys. Acta. 1838, 1586–1593. 

Jaks and Stats are critical components of many cytokine receptor systems; regulating growth, survival, differentiation, and pathogen resistance. An example of these pathways 

is shown for the IL-6 (or gp130) family of receptors, which coregulate B cell differentiation, plasmacytogenesis, and the acute phase reaction. Cytokine binding induces 

receptor dimerization, activating the associated Jaks, which phosphorylate themselves and the receptor. The phosphorylated sites on the receptor and Jaks serve as docking 

sites for the SH2-containing Stats, such as Stat3, and for SH2-containing proteins and adaptors that link the receptor to MAP kinase, PI3K/Akt, and other cellular pathways. 

Phosphorylated Stats dimerize and translocate into the nucleus to regulate target gene transcription. Members of the suppressor of cytokine signaling (SOCS) family dampen 

receptor signaling via homologous or heterologous feedback regulation. Jaks or Stats can also participate in signaling through other receptor classes, as outlined in the Jak/ 

Stat Utilization Table. Researchers have found Stat3 and Stat5 to be constitutively activated by tyrosine kinases other than Jaks in several solid tumors. 

The Jak/Stat pathway mediates the effects of cytokines, like erythropoietin, thrombopoietin, and G-CSF, which are protein drugs for the treatment of anemia, thrombocytopenia, 

and neutropenia, respectively. The pathway also mediates signaling by interferons, which are used as antiviral and antiproliferative agents. Researchers have found that 

dysregulated cytokine signaling contributes to cancer. Aberrant IL-6 signaling contributes to the pathogenesis of autoimmune diseases, inflammation, and cancers such as 

prostate cancer and multiple myeloma. Jak inhibitors currently are being tested in models of multiple myeloma. Stat3 can act as an oncogene and is constitutively active in 

many tumors. Crosstalk between cytokine signaling and EGFR family members is seen in some cancer cells. Research has shown that in glioblastoma cells overexpressing 

EGFR, resistance to EGFR kinase inhibitors is induced by Jak2 binding to EGFR via the FERM domain of the former [Sci. Signal. (2013) 6, ra55]. 

Activating Jak mutations are major molecular events in human hematological malignancies. Researchers have found a unique somatic mutation in the Jak2 pseudokinase 

domain (V617F) that commonly occurs in polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. This mutation results in the pathologic activation Jak2, 

associated with receptors for erythropoietin, thrombopoietin, and G-CSF, which control erythroid, megakaryocytic, and granulocytic proliferation and differentiation. Researchers 

have also shown that somatic acquired gain-of-function mutations of Jak1 are found in adult T cell acute lymphoblastic leukemia. Somatic activating mutations in Jak1, 

Jak2, and Jak3 have also been identified in pediatric acute lymphoblastic leukemia (ALL). Furthermore, Jak2 mutations have been detected around pseudokinase domain 

R683 (R683G or DIREED) in Down syndrome childhood B-ALL and pediatric B-ALL. 


Beekman, R. and Touw, I.P. (2010) Blood 115, 5131–5136. • Neurath, M.F. and Finotto, S. (2011) Cytokine Growth Factor Rev. 22, 83–89. • Sansone, P. and Bromberg, 

J. (2012) J. Clin. Oncol. 30, 1005–1014. • Vainchenker, W. and Constantinescu, S.N. (2013) Oncogene 32, 2601–2613. • Yu, H., Pardoll, D., and Jove, R. (2009) Nat. 

Rev. Cancer 9, 798–809. 

© 2009–2015 Cell Signaling Technology, Inc. • We would like to thank Dr. Pranoti Mandrekar, University of Massachusetts Medical School, Worcester, MA, for reviewing this diagram. 


© 2002–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Stefan Constantinescu, Ludwig Institute for Cancer Research, Brussels, Belgium for reviewing this diagram. 




NF-κB Signaling 

Stress: ROIs, 

UV, metals, 

ischemia, shear 

UV 

Ag 

BCR 

JNK 

p38 

ub K63-ubiquitin 

ub K48-ubiquitin 

Cytoplasm 

Nucleus 

Ag-MHC 

TCR 

For detailed signaling, 

see BCR Pathway. 

CK2 

RelA/cRel IκBα/ε 

NF-κB1 

p50 

LPS, CpG, 

ssRNA, dsRNA 

TLRs 

IκBζ 

Pellino 

ub 

RelA/cRel IκBα/β/ε 

NF-κB1 

p50 

ub 

Bcl-3 

NF-κB 

p50/52 

NF-κB 

p50/52 

ub 

NAP1 NAK 

RSK1 

CYLD 

p65/ 

RelA 

NF-κB 

p50/52 

ac 

IL-1 

IL-1R 

MyD88 

ub IRAK1/4 

TRAF2/6 

c-IAP1/2 

For detailed signaling, 

see TLR Pathway. 

Ubc13 

TRAF6 

A20 

For detailed signaling, UEV1A 

ITCH TAX1BP1 

ub 

see TCR Pathway. 

ub 

RNF11 

TRAF6 

TAB1/2 

TAK1 

LUBAC 

ELKS 

HOIL1 HOIP 

IKKβ IKKα 

SHARPIN 

IKKγ/ 

NEMO 

ub 

ub 

ub 

β-TrCP 

Nuclear-cytoplasmic 

shuttling of nonphosphorylated 

forms 

Proteasomal 

Degradation 

IKKα/β/ε 

PKCζ 

PKA C 

p65/ 

RelA 

NF-κB 

p50/52 

PCAF 

CBP/ 

p300 

HDAC 

Survival, Proliferation, Inflammation, Immune Regulation 

TNFR 

TRADD 

TRAF2/5 

RIP 

ub 

Tax 

ub 

p65/ 

IκBα RelA 

NF-κB2 

p52 

GSK-3β 

CK2 

SUMO 

SUMO 

IKKγ/ 

NEMO 

PIASγ 

MSK1 

ATM 

Growth Factors: 

BMP, EGF, HGH, 

Insulin, NGF, TGF-α 

ub 

ubc13 

Akt 

Cot 

ub 

Genotoxic 

Stress 

IKKγ/ 

NEMO 

PARP1 

GF-Rs 

Ras 

PDK1 

β-TrCP 

IKKα 

IKKα 

PI3K 

H3 

ub 

ub 

IKKα 

IKKα 

TRAF2 

c-IAP1/2 

IKKα 

LT, CD40L, 

BAFF/BLys 

LTβR, 

CD40, 

BR3 

NIK 

NF-κB2 

p100 

NF-κB2 

p52 

NF-κB2 

p52 

TRAF3 

IKKα 

Proteasomal 

Processing 

RelB 

RelB 

RelB 

Lymphogenesis, B Cell Maturation 

Nuclear factor-κB (NF-κB)/Rel proteins include NF-κB2 p52/p100, NF-κB1 p50/p105, c-Rel, RelA/p65, and RelB. These proteins function as dimeric transcription factors that 

regulate the expression of genes influencing a broad range of biological processes including innate and adaptive immunity, inflammation, stress responses, B-cell development, 

and lymphoid organogenesis. In the classical (or canonical) pathway, NF-κB/Rel proteins are bound and inhibited by IκB proteins. Proinflammatory cytokines, LPS, growth 

factors, and antigen receptors activate an IKK complex (IKKβ, IKKα, and NEMO), which phosphorylates IκB proteins. Phosphorylation of IκB leads to its ubiquitination and 

proteasomal degradation, freeing NF-κB/Rel complexes. Active NF-κB/Rel complexes are further activated by post-translational modifications (phosphorylation, acetylation, 

glycosylation) and translocate to the nucleus where, either alone or in combination with other transcription factors including AP-1, Ets, and Stat, they induce target gene 

expression. In the alternative (or noncanonical) NF-κB pathway, NF-κB2 p100/RelB complexes are inactive in the cytoplasm. Signaling through a subset of receptors, including 

LTβR, CD40, and BR3, activates the kinase NIK, which in turn activates IKKα complexes that phosphorylate C-terminal residues in NF-κB2 p100. Phosphorylation of NF-κB2 

p100 leads to its ubiquitination and proteasomal processing to NF-κB2 p52. This creates transcriptionally competent NF-κB p52/RelB complexes that translocate to the 

nucleus and induce target gene expression. Only a subset of NF-κB agonists and target genes are shown here. 


Gilmore T.D. (2014) www.nf-kb.org • Hayden M.S. and Ghosh S. (2008) Cell 132, 344–362. • Perkins N.D. (2006) Oncogene 25, 6717–30. • Sun S-C. (2012) Immunol 

Rev. 246, 125–140. • Chen J. and Chen Z.J. (2013) Curr. Opin. Immunol. 25, 4–12. 

TNF 

CYLD 

Tumor Immunology 

MMPs 

VEGF 

T cell apoptosis 

IL-10 

IDO 

DC 

T Reg 

FoxP3 

CD4 + 

IL-10 

TGF-β 

IL-35 

Th1 

T-bet 

TNF-α 

IL-2 

CCL22 

MMPs 

VEGF 

Angiogenesis 

IFNγ 

T cell 

Immune 

Checkpoint 

MSC 

Arginase 

IL-10 

TGF-β 

IFNγ-R 

CTL 

T-bet 

T cell 

priming 

IL-1β 

TNF-α 

CD4 + 

T cell 

Tumor-draining Lymph Node 

Proliferation and production 

of anti-tumor antibodies 

Jak3 

Stat3 

cytotoxic 

granules 

Stat1 

B cell 

IL-2, 4, 5 

Tumor-specific 

CD8 + T cell 

TIM-3 

Galectin-9 

 

B7-H3 

Class I antigen 

presentation; 

IDO; PD-L1 

Activation/Response Change 

 

B7-H4 

Genes of cell growth and survival; 

PD-L1, VEGF, IL-6, IL-10 

PD-1 

PD-L1 

Nucleus 

NF-κB 

TCR 

MHC 

Stat3 

Akt 

MAPK 

DC 

M2 

Macrophage 

IFNγ 

cytotoxic 

granules 

TNF-R 

oncogenic 

signaling 

IL-6 

NK 

T-bet 

Jak 

TNF-α 

IL-1β 

Tumor-promoting 

Macrophage 

IL-10 

TGF-β 

M1 

Macrophage 

IL-6R 

Tumor Cell 

MMPs 

IL-4 

CCL22 

Inflammation 

IL-1β 

TNF-α 

ALK 

ROS1 

RTK: EGFR 

HER2 

etc. 

Mast cell 

Tumor cells employ multiple defense strategies to evade detection by the immune system. One common strategy, upregulation of immune checkpoint proteins and ligands, 

takes advantage of a natural immune mechanism for self-tolerance and prevention of collateral tissue damage. Immune checkpoint receptors, such as PD-1, CTLA-4, and 

many others, are located on T cells and engage with their corresponding ligand on tumor cells and dendritic cells, sending inhibitory signals that repress T cell activation 

or response. One of the first discovered checkpoint proteins, CTLA-4, plays a role at the stage of T cell priming by binding to the CD28 ligands CD80 or CD86 to prevent 

co-stimulatory signals necessary for T cell activation. In contrast, the PD-1/PD-L1 checkpoint acts later in the process, inhibiting anti-tumor immune responses by effector 

T cells such as CD4 + T helper 1 (Th1) cells and CD8 + cytotoxic T lymphocytes (CTLs), leading to decreases in IFNγ production and cytolytic activity. Upregulation of PD-L1 

expression on the tumor cell surface is mediated by IFNγR signaling to Stat1, as well as oncogenic signaling through several receptor tyrosine kinases (EGFR, ALK, ROS, 

HER2, and others) to activate the MAPK, Akt, and Stat3 pathways. 

Cells in the tumor microenvironment can also influence tumor progression. FoxP3 + /CD4 + T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs) secrete immunosuppressive 

cytokines IL-10 and TGF-β to inhibit the activity of Th1 cells and CTLs. Natural killer (NK) cells release cytotoxic granules against the tumor cell and secrete 

IFNγ, which stimulates surrounding pro-inflammatory M1 macrophages. Pro-tumorigenic M2 macrophages suppress anti-tumor immune responses via production of IL-10 

and TGF-β and promote metastasis through release of MMPs. MMPs and TGF-β are also released by surrounding mast cells. 


Pardoll, D.M. (2012) Nat. Rev. Cancer 12, 252–264. • Vanneman, M. and Dranoff, G. (2012) Nat. Rev. Cancer 12, 237–251. • Kawakami, Y., Yaguchi, T., and Park, J.H., 

et al. (2013) Front. Oncol. 3, 136. • Elinav, E., Nowarski, R., Thaiss, C.A., et al. (2013) Nat. Rev. Cancer 13, 759–771. • Mentlik, J.A., Cohen, A.D., and Campbell, K.S. 

(2013) Front. Immunol. 4, 481. • Gajewski, T.F., Schreiber, H., and Fu, Y.X. (2013) Nat. Immunol. 14, 1014–1022. • Krstic, J. and Santibanez, J.F. (2014) ScientificWorld- 

Journal, 521754. 

MMPs 

TGF-β 

Th2 

IL-13 

© 2009–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Thomas D. Gilmore, Boston University, Boston, MA, for reviewing this diagram. 


© 2014–2015 Cell Signaling Technology, Inc. • We would like to thank Glenn Dranoff, M.D., Susanne H.C. Baumeister, M.D., 

Karrie Wong, Ph.D., and Girija Goyal, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, for reviewing this diagram. 


08 


Neuronal 

Development Markers 

Neural progenitor: Nestin, Musashi-1 

Astroglial precursor: Notch1 

Oligodendrocyte progenitor: A2B5, 

PDGFRα 

Neuronal differentiation: CEND1 

Neuronal stem cell marker: Sox1, Sox2 

Neuron: Neurofilament L, Neurofilament 

M, Neurofilament H, β3-tubulin, 

MAP2, Tau 

Oligodendrocytes: CNPase, MAG, MBP 

Schwann: Vimentin 

Astroglia: GFAP 

Neuroscience 

Neuroscience is a broad scientific area consisting of cellular and molecular biology, anatomy, physiology, 

and development of neurons and the nervous system. The field also includes cognitive neuroscience 

and behavioral research. 

Neuronal Development 

Development of the peripheral and central nervous systems begins early in embryogenesis and can 

be tracked throughout its different stages using lineage markers specific to each stage of neuronal 

development. Neural stem cells are derived from the ectoderm and differentiate into neural crest cells, 

glial progenitor cells, and neuronal progenitor cells. Markers for neural stem cells include Sox1 and 

Sox2. The neural crest further differentiates into a diverse array of cell types including neurons, glia, 

craniofacial cartilage, and connective tissue, and is sometimes referred to as the fourth primary germ 

layer. Neural crest markers include FoxD3 and Notch1. Glial progenitor cells develop into astrocytes, 

which provide structural support and help form the blood-brain barrier, and oligodendrocytes, which 

form the insulating myelin sheaths that surround axons. Neuronal progenitor cells, which can be identified 

using the markers Nestin and Musashi-1, give rise to mature neurons. 

Sox1, a marker for neuronal stem cells, is expressed 

in 1-day old rat brain but not adult rat brain. 

Sox1 Antibody #4194: Confocal IF 

analysis of postnatal day 1 (left) and 

adult (right) rat brain using #4194 (green) 

and Neurofilament-L (DA2) Mouse 

mAb #2835 (red). Blue pseudocolor = 


Synaptic signaling occurs when the signal from one neuron is transmitted across the synaptic cleft 

to another neuron through the action of neurotransmitters (NTs). These NTs, such as dopamine, 

glutamate, and GABA (γ-aminobutyric acid), are stored in synaptic vesicles in the presynaptic neuron. 

Upon receiving an action potential, vesicles containing NT dock, prime, and fuse to the presynaptic 

membrane in a highly regulated mechanism through the action of SNARE family (VAMP, syntaxin-1, 

SNAP25), chaperone (complexin), and calcium binding proteins (synaptotagmin). Vesicle fusion results 

in NT release into the synaptic cleft, where it binds one of several receptors on the postsynaptic 

membrane. Receptor families such as the dopamine receptor, which is a GPCR, can signal through 

adenylate cyclase to activate PKA and other signaling intermediates to regulate gene expression 

through the actions of CREB and other transcription factors. Other NTs bind ion channels such as 

NMDAR or AMPAR that regulate flux of Ca 2+ and Na + , thereby perpetuating the action potential through 

the postsynaptic neuron. Continual NT release into a synapse and clustering of postsynaptic receptors 

can strengthen synaptic signaling over time. Synaptic plasticity, or the ability to modulate the number 

of NT receptors at the synapse, is a mechanism involved in many adaptative processes such as stress, 

addiction, and learning and memory. 

VAMP2, a SNARE 

protein expressed in 

brain tissue, cell lines, 

and primary neurons, 

facilitates the docking, 

priming, and fusion of 

NT-containing vesicles 

to the presynaptic 

membrane. 

A 

Neuronal Markers 

Markers are proteins with a very specific and well-defined localization that are used to identify or localize 

a subset of neurons (i.e. glutamatergic or GABAergic) or cellular compartment (i.e. presynaptic or 

postsynaptic compartment). 

B 

kDa 

60 

50 

40 

30 

20 

10 

1 2 3 4 5 6 

VAMP2 

chapter 08: Neuroscience 

VAMP2 (D6O1A) Rabbit mAb #13508: 

Confocal IF analysis of primary rat cortical 

neurons grown for 21 days (A) using 



WB analysis of extracts from various cell 

lines and tissues (B) using #13508. 

Lanes 


2. rat brain 

3. human cerebellum 

4. OVCAR8 

5. SK-N-SH 

6. Neuro-2a 

Synaptic Signaling 

Neurons are the building blocks of extensive neural networks. Transfer of information between neurons 

occurs at the synapse, where the neuronal information is converted from electrical action potentials 

into neurochemical signals. The synapse comprises a presynaptic active zone, the synaptic cleft, and 

the postsynaptic density. 

Neuronal Type 

Glutamatergic: EAAT1, EAAT2, EAAT3, VGluT1, VGluT2 

Dopaminergic: Tyrosine hydroxylase, Parkin 

GABAergic: GAD1, GAD2, DARPP-32 

Subcellular Compartment 

Presynaptic: Synapsin-1, Synaptophysin, SYT1, NSF 

Postsynaptic: PSD95, SHANK2 

Dentrite: MAP2 

Axon: β3-tubulin, Tau 

PSD95, a scaffolding 

protein within the 

postsynaptic density, is 

expressed in rat retina. 

Synaptic vesicle 

Voltage-gated 

Ca 2+ channels 

Presynaptic 

Active Zone 

Synapsin-1 (D12G5) XP ® Rabbit mAb 


brain (left) or primary rat cortical neurons 

grown for 21 days (right) using #5297 

(green) and β3-Tubulin (TU-20) Mouse 

mAb #4466 (red). Blue pseudocolor = 


Synapsin-1, a 

presynaptic marker, 

is expressed in 

mouse brain and 

primary neurons. 

PSD95 (D27E11) XP ® Rabbit mAb 

#3450: Confocal IF analysis of rat retina 

using #3450 (red) and Neurofilament-L 

(DA2) Mouse mAb #2835 (green). Blue 


DNA dye). 

Neurotransmitter 

Neurotransmitter 

receptors 

Synaptic 

Cleft 

Post-synaptic 

density 

MAP2 (D5G1) XP ® Rabbit mAb #8707: 

Confocal IF analysis of frozen rat cerebellum 

(left) or primary rat cortical neurons 

grown for 21 days (right) using #8707 



MAP2, a marker 

for dendrites, is 

expressed in rat 

cerebellum and 

primary neurons. 


www.cellsignal.com/cstneuroscience 185


α-Synuclein is the 

main component of 

pathogenic Lewy 

bodies and Lewy 

neurites characteristic 

of Parkinson’s 

disease. 

Rev-Erbα regulates 

expression of several 

genes, including 

circadian regulator 

protein BMAL1. 


Devastating diseases arise from loss of neuron structure or function and are generally known as neurodegenerative 

diseases. One of the most common neurodegenerative diseases worldwide is Alzheimer’s 

disease (AD). This condition is characterized by the presence of extracellular amyloid plaques that form 

through abnormal APP (amyloid β precursor protein) processing and aggregation of β-amyloid peptides. 

AD is also characterized by the formation of neurofibrillary tangles that result from hyperphosphoryation 

of the tau protein. Parkinson’s disease, another neurodegenerative disorder, occurs when genetic mutation 

or environmental toxins result in misfolded α-synuclein protein that aggregates to form Lewy bodies. 

These aggregates alter dopamine signaling, particularly in the nigrostriatal pathway, ultimately leading 

to neuronal dysfunction and cell death. 


0.007 

0.006 

0.005 

0.004 

0.003 

0.002 

0.001 

0 

A 

BMAL1 NR1D1 α Satellite 

β-amyloid fragments aggregate 

to form the amyloid plaques 

characteristic of Alzheimer’s disease. 

β-Amyloid (D54D2) XP ® Rabbit mAb #8243: Confocal IF analysis of paraffin-embedded 

human Alzheimer’s brain using #8243 (green) and Tau (Tau46) Mouse mAb #4019 (red). 


Circadian Rhythms 

Circadian rhythms govern many key physiological processes that fluctuate with a period of approximately 

24 hours. These processes include the sleep-wake cycle, glucose, lipid and drug metabolism, heart 

rate, hormone secretion, renal blood flow, and body temperature, as well as basic cellular processes 

such as DNA repair and the timing of the cell division cycle. The mammalian circadian system consists 

of many individual tissue-specific clocks (peripheral clocks) that are controlled by a master circadian 

pacemaker residing in the suprachiasmatic nuclei (SCN) of the brain. The periodic circadian rhythm is 

prominently manifested by the light-dark cycle, which is sensed by the visual system and processed by 

the SCN. The cellular circadian clockwork consists of interwoven positive and negative regulatory loops, 

or limbs. The positive limb includes the CLOCK and BMAL1 proteins, two transcription factors that bind 

E box enhancer elements and activate transcription of their target genes. The negative limb is formed 

by CRY and PER proteins, which inhibit CLOCK/BMAL1-mediated transcriptional activation. In tissues, 

roughly six to eight percent of all genes exhibit a circadian expression pattern. For example, expression 

of the nuclear receptor Rev-Erbα oscillates with circadian rhythm in liver cells. Rev-Erbα regulates 

expression of several key regulators of circadian rhythm, including BMAL1, ApoA-I, and ApoC-III. 


B 

Rev-Erbα (E1Y6D) Rabbit mAb #13418 


α-Synuclein (D37A6) XP ® Rabbit 

mAb #4179: IHC analysis of paraffinembedded 

mouse brain (A) using 

#4179. Confocal IF analysis of normal 

rat cerebellum using #4179 (green) (B). 



Rev-Erbα (E1Y6D) Rabbit mAb #13418: Chromatin IPs were performed with 

cross-linked chromatin from 4 x 10 6 Hep G2 cells and either 10 μl of #13418 

or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP ® Enzymatic Chromatin IP 

Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR 

using human BMAL1 promoter primers, SimpleChIP ® Human NR1D1 Promoter 

Primers #13413, and SimpleChIP ® Human α Satellite Repeat Primers #4486. 

The amount of immunoprecipitated DNA in each sample is represented as a 

percent of the total input chromatin. 


Dzamko, N., Zhou, J., Huang, Y., and Halliday, G.M. (2014) Front. Mol. Neurosci. 7, 57. • Florio, M. and Huttner, W.B. (2014) 

Development 141, 2182−2194. • Franco, S.J. and Müller, U. (2013) Neuron 77, 19−34. • Kalsbeek, A., la Fleur, S., and 

Fliers, E. (2014) Mol. Metab. 3, 372−383. • Kojetin, D.J. and Burris, T.P. (2014) Nat. Rev. Drug Discov. 13, 197−216. • 

Spires-Jones, T.L. and Hyman, B.T. (2014) Neuron 82, 756−771. • Südhof, T.C. (2013) Neuron 80, 675−690. • Südhof, T.C. 

(2013) Nat. Med. 19, 1227−1231. • Tenreiro, S., Eckermann, K., and Outeiro, T.F. (2014) Front. Mol. Neurosci. 7, 42. 

Commonly Studied Neuroscience Targets 

Target M P 

A2B5 

• 

AMPA Receptor (GluR 1) • 

• 

Phospho-AMPA Receptor 

(GluR 1) (Ser845) 

AMPA Receptor (GluR 2/3/4) 

AMPA Receptor (GluR 2) 


(GluR 2) (Tyr869/873/876) 


(GluR 2) (Tyr876) 



(Arg860) 

APBA2 

ApoE 

ApoE4 

APP 

Phospho-APP (Thr668) 

APP/β-Amyloid 

β-Amyloid 

Arrestin 1/S-Arrestin 

Ataxin-1 

BACE 

β-Amyloid (pE3 Peptide) 

β-Amyloid (1-37 Specific) 




BMAL1 

Brn2/POU3F2 

BRSK1 

BRSK2 

Bassoon 

Calbindin 

CaMKI-δ 

CaMKII-α 

CaMKII (pan) 

Phospho-CaMKII (Tyr231) 

Phospho-CaMKII (Thr286) 

CaMKIV 

Phospho-CAMKK2 (Ser511) 

CASK 

Caspr2 

Cathepsin B 

CD13/APN 

CDK5 

CEND1 

CIRBP 

CK1δ 

CK1ε 

CLCN3 

• Phospho-CREB (Ser133) 

• CRMP-2 

• Phospho-CRMP-2 (Thr514) 

Dab1 

• 

• • 

• • 

• 

• 

• 

• 

• • 

• 

• • 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• • 

• • 

• 

• • 

• 

• 

• • 

• 

• 

• 

• • 

• • 

• 

• 

• 

• 

Target M P 

CNPase 

• • 

Complexin-1 

Complexin-1/2 

CREB 

Phospho-Dab1 (Tyr220) 

Phospho-Dab1 (Tyr232) 

DAG Lipase α 

DARPP-32 

Phospho-DARPP-32 (Thr34) 

Phospho-DARPP-32 (Thr75) 

Phospho-DARPP-32 (Ser97) 

DCBLD2 

DDC 

Delta FosB 

DJ-1 

Dopamine β-Hydroxylase 

(DBH) 

Doublecortin 

Phospho-Doublecortin 

(Ser297) 

Phospho-Doublecortin 

(Ser334) 

Drebrin 

Drebrin A 

DYRK1A 

Dyrk1B 

Dysbindin 

EAAT1 

EAAT2 

EAAT3 

EGR1 

EGR3 

FABP7 

FE65 

GABA(B)R1 

GABA(B)R2 

GAD1 

GAD2 

GAP43 

GFAP 

GGA3 

Cleaved GGA3 (Asp313) 

GKAP 

Glutamate Dehydrogenase 

1/2 

GNB3 

GPR50 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• 

• 

• 

• Munc18-1 

• Musashi 

Myelin Basic Protein 

• 

• 

• 

• • 

• • 

• 

• • 

• 

• 

• 

• 

• 

• 

• 

• • 

• 

• • 

• • 

• 

• • 

• 

• 

• 

• 

• • 

Target M P 

GRAF1 

GRK2 

GSTP1 

Homer1 

5-HTR1A 

5-HTR4 

Huntingtin 

KISS1R 

LIS1 

LRRK2 

MAG 

MAP2 

Phospho-MAP2 (Ser136) 

Phospho-MAP2 

(Thr1620/1623) 

MELK 

Mena 

Merlin 

Phospho-Merlin (Ser518) 

mGluR1 

mGluR2 

STOP 

Na Channel β1 Subunit 

NCS1 

Nestin 

NeuN 

NeuroD 

Neurofilament-H 

Neurofilament-L 

Neurofilament-M 

Neurogenin 2 

Neuropeptide Y 

Neuropilin-1 

Neuropilin-2 

NG2 

NGF 

NHERF1 

NHERF2 

Nicastrin 

NKCC1 

NMDAR1 

Phospho-NMDAR1 (Ser890) 



NMDAR2A 

Phospho-NMDAR2A 

(Tyr1246) 

NMDAR2B 

• 

• 

• 

• 

• 

• 

• • 

• 

• 

• • 

• • 

• • 

• 

• 

• 

• • 

• 

• • 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• 

• 

• 

• 

• • 

• 

• • 

• • 

• 

• 

• 

• 

• 

• 

• • 



nodes within neuroscience pathways 

and are commonly studied in 

neuroscience research. Primary 








www.cellsignal.com/cstneuroscience 187

SYT SYT 

VAMP VAMP 

SYT SYT 

VAMP VAMP 

VAMP 

Syntaxin 1 

Syntaxin 1 

Dynamin 

Syntaxin 1 



75 


CST antibodies for Phospho-CREB 




Target M P 

Phospho-NMDAR2B • 

(Tyr1070) 


(Ser1284) 


(Tyr1472) 

Nna1 

• 

nNOS 

• • 

Nogo-A 

• 

NT5E/CD73 • 

Oligophrenin-1 • 

Phospho-µ-Opioid Receptor • 

(Ser375) 

p35/25 • 

p39 

• 

p75NTR 

• • 

PARK9 

• 

Parkin 

• • 

PC1/3 

• 

PC2 

• 

PEN2 

• • 

PINK1 

• 

Plexin A1 

• 

Plexin A2 • • 

Plexin A3 • 

Plexin A4 • 

Presenilin-1 • • 

Presenilin-2 • • 

PSD93 

• 

Phospho-PSD93 (Tyr340) • 

PSD95 

• • 

Phospho-PSD95 • 

(Tyr236/Tyr240) 

Target M P 

Ras-GRF1 

Phospho-Ras-GRF1 (Ser916) 

RGS4 

Rhodopsin 

SAP102 

Secretagogin 

Semaphorin 3B 

Phospho-Semaphorin 4B 

(Ser825) 


Madiraju, A.K. et al. (2014) Metformin suppresses gluconeogenesis 

by inhibiting mitochondrial glycerophosphate 

dehydrogenase. Nature 510, 542–546. 

Villeda, S.A. et al. (2014) Young blood reverses age-related 

impairments in cognitive function and synaptic plasticity in 

mice. Nat. Med. 20, 659–663. 



16, 99–107. 

Kawai, M. et al. (2014) Sympathetic activation induces 

skeletal Fgf23 expression in a circadian rhythm-dependent 

manner. J. Biol. Chem. 289, 1457–1466. 

Parisiadou, L. et al. (2014) LRRK2 regulates synaptogenesis 

and dopamine receptor activation through modulation of PKA 

activity. Nat. Neurosci. 17, 367–376. 

• 

• 

• 

• 

• 

• 

• 

• 

SHANK2 

• 

Shootin1 

• 

SLC1A4 

• 

SNAP25 

• • 

SOD1 

• • 

Spinophilin • • 

SSTR1 

• 

Stargazin • • 

STEP 

• • 

Non-phospho-STEP (Ser221) • 

Synapsin 

• • 

Phospho-Synapsin (Ser9) • 

Synaptophysin • • 

SynGAP 

• • 

Syntaxin 1A 

• 

α-Synuclein • • 

α/β-Synuclein • 

Synaptotagmin • • 

Tau 

• 

Phospho-Tau (Thr181) • 

Phospho-Tau (Ser202) • 

Phospho-Tau (Ser396) • 

Target M P 

• 

Phospho-Tau (Ser400/ 

Thr403/Ser404) 

TDP43 

Tenascin C 

TFAM 

Thy1 

TMP21 

Torsin A 

TPH-1 

Trk (pan) 

TrkA 

Phospho-TrkA (Tyr490) 


/TrkB (Tyr516) 

Phospho-TrkA (Tyr674/675) 

/TrkB (Tyr706/707) 


/TrkB (Tyr816) 

TrkB 

TrkC 

β3-Tubulin 

Tyrosine Hydroxylase 

Phospho-Tyrosine 

Hydroxylase (Ser31) 

Phospho-Tyrosine 

Hydroxylase (Ser40) 

UNC5B 

VAMP2 

VAMP3 

VGLUT1 

VGLUT2 

Vti1a 

• 

• 

• • 

• 

• 

• 

• 

• 

• • 

• 

• 

• 

• 

• • 

• 

• 

• 

• • 

• 

• 

• 

• 

• 

• • 

• 

Azeloglu, E.U. et al. (2014) Interconnected network motifs 

control podocyte morphology and kidney function. Sci Signal. 

7, ra12. 

Ma, L. et al. (2014) Cluster of differentiation 166 (CD166) 

regulated by phosphatidylinositide 3-Kinase (PI3K)/AKT signaling 

to exert its anti-apoptotic role via yes-associated protein 

(YAP) in liver cancer. J. Biol. Chem. 289, 6921–6933. 

Socodato, R. et al. (2014) The nitric oxide-cGKII system 

relays death and survival signals during embryonic retinal 

development via AKT-induced CREB1 activation. Cell Death 

Differ. 21, 915–928. 

Liu, J. et al. (2014) Insulin-like growth factor-1 and bone 

morphogenetic protein-2 jointly mediate prostaglandin E2- 

induced adipogenic differentiation of rat tendon stem cells. 


Antibody Validation Principles 

Please visit our website to learn more about what Antibody Validation means at Cell Signaling Technology. 


Vesicle Trafficking in Presynaptic Neurons: Synchronous Release 

Ca 2+ 

Channel 

Ca 2+ 

Channel 

SYT SYT 

VAMP VAMP 

SYT SYT 

VAMP VAMP 

SYT1 

Munc13 

RIM 

RIM-BP 

SYT SYT 

VAMP VAMP 

VAMP 

SYT SYT 

VAMP VAMP 

Munc13 

RIM 

RIM-BP 

Rab 

SYT1 

SYT1 

VAMP 

A 

Clathrin 

Docking 

Rab 

(NT) 

Munc18-1 

AP180 

AP-2 

SYT1 

SYT1 

VAMP 

C 

Super 

Priming 

(NT) 

SNAP25 

Complexin 

SYT1 

Amphiphysin 

Munc18-1 

SNAP25 

Vesicle 

Recycling 

Ca 2+ 

Ca 2+ 

Channel 

Ca 2+ Ca 2+ 

Munc13 

RIM 

RIM-BP 

Rab 

Postsynaptic Neuron 

Ca 2+ 

Channel 

Munc13 

RIM 

RIM-BP 

D 

Fusion 

and 

Release 

(NT) 

B 

Priming 

Rab 

(NT) 

VAMP 

VAMP 

SNAP25 

Ca 2+ 

SYT1 

SNAP25 

Ca 2+ 

SYT1 

Munc18-1 

Syntaxin 1 

Munc18-1 

Complexin 

SERT 

EAAT 

DAT 

NT 

Re-uptake 

Neuronal communication is a very connective process. Transfer of information between neurons occurs at the synapse, where the neuronal information is converted from 

electrical action potentials into neurochemical signals. The synapse comprises a presynaptic active zone (a clustering of vesicle fusion sites and calcium channels on the 

presynaptic cell membrane), the synaptic cleft, and the postsynaptic density, an electron-dense domain of the postsynaptic neuron specializing in the reception and integration 

of synaptic signals. Intracellular vesicles containing neurotransmitter (NT) rapidly fuse to the presynaptic membrane and release their contents into the synaptic cleft upon 

arrival of an action potential—a type of neurotransmission termed synchronous release. The docking, priming, and fusion of these vesicles is carried out by SNARE family and 

other chaperone proteins located on both the vesicle and presynaptic cell membrane. Synaptic vesicles dock to predetermined sites in the active zone through the interaction 

of vesicle-associated Rab3 (or Rab27) with RIM, which can bind to calcium channels directly and via RIM-BP (A). SNARE proteins might also play a role in docking based on 

studies of non-neuronal cells, but there is no conclusive evidence for such a role in mammalian neurons. The vesicle SNARE protein, VAMP (also called synaptobrevin), binds 

to SNARE proteins on the cell membrane, syntaxin 1 and SNAP25, priming the vesicle for fusion (B). Munc18-1 binds to monomeric syntaxin 1 as well the SNARE complex 

and assists with complex assembly. The co-chaperone protein complexin and the calcium-binding protein synaptotagmin 1 (SYT1) associate with SNARE proteins to form tight 

complexes, bringing the lipid membranes together (C). When an action potential in the presynaptic neuron opens voltage-gated calcium channels, calcium binds to SYT1 

and allows SYT1 to interact with the SNARE complex as well as the plasma membrane resulting in membrane fusion and release of NT into the synaptic cleft (D). The fast 

response to an action potential is due in part to the proteins RIM, RIM-BP, and Munc13, which form physical interactions between the vesicle, the cell membrane, and calcium 

channels, bringing the three necessary elements into close proximity. Released NT can be recycled through specific transporters such as EAATs (reuptake of glutamate) or a 

monoamine transporter, such as SERT (reuptake of serotonin) or DAT (reuptake of dopamine) back into the cytoplasm of the neuron. 


Blakely, R.D. and Edwards, R.H. (2012) Cold Spring Harb. Perspect. Biol. 4, a005595. • Jahn, R. and Fasshauer, D. (2012) Nature 490, 201–207. • Saheki, Y. and De 

Camilli, P. (2012) Cold Spring Harb. Perspect. Biol. 4, a005645. • Südhof, T.C. (2013) Neuron 80, 675−690. • Südhof, T.C. (2013) Nat. Med. 19, 1227–1231. • Südhof, 

T.C. (2012) Neuron 75, 11–25. 

© 2015 Cell Signaling Technology, Inc. • We would like to thank Taulant Bacaj, Stanford School of Medicine, Palo Alto, CA for reviewing this diagram. 



189

II 

Antibody 

Applications 

Studies of cellular regulation may employ a variety of immunodetection 

methods, with sample types spanning cultured cells, primary cells, 

and tissue samples and their lysates. Using multiple applications for 

investigation provides valuable complementary information and confirms 

data validity. The insights gained can, in turn, help you communicate a 

more comprehensive understanding of cellular signaling mechanisms to 

the research community. In this context it is important to understand the 

strengths and limitations of each method. At Cell Signaling Technology, 

it is our philosophy to share all our methods and tips that help ensure 

successful experiments. In this section of the CST Guide, we discuss 

and illustrate best practice approaches to overcoming obstacles commonly 

faced when performing antibody-based applications. 


Tips for Success 

Application Protocols 

Troubleshooting Guides 


Key components of protein synthesis, including transcription and translational machinery, 

are illustrated at the junction between the nuclear pore and endoplasmic reticulum. 


193

09 

Section II: ANTIBODY APPLICATIONS 

Chromatin Immunoprecipitation 

(ChIP) 

Optimal antibody concentration is critical for efficient ChIP. 

Using either too much or too little antibody can result in significantly lower enrichment, potentially leading 

to difficulty in detecting a low frequency, low stability binding event. Shown below are antibody titration 

data generated as part of CST’s rigorous ChIP validation process for p53 (1C12) Mouse mAb #2524. 

chapter 09: Chromatin Immunoprecipitation (ChIP) 

The ChIP assay is a powerful and versatile technique used for probing protein-DNA interactions within 

the natural chromatin context of the cell. The ChIP procedure isolates protein-DNA complexes that have 

been chemically crosslinked and harvested, using an antibody to enrich for specific protein associations 

by immunoprecipitation. After reversing the crosslinks, the DNA associated with a particular protein 

can be analyzed by PCR or ChIP-Seq. 

The success or failure of a ChIP experiment is highly dependent on the integrity of the chromatin, the 

quality of the epitope, and the specificity of the antibody. This is especially true for low abundance, 

low stability interactions—such as the binding of transcription factors (e.g., TCF4) and cofactors (e.g., 

Ezh2) that may fall under the detection threshold if the integrity of the protein or DNA is compromised, 

or if the immunoenrichment relies on an antibody that is not highly specific to the target of interest. 

ChIP Tips for Success 

Enzymatic digestion better preserves chromatin integrity. 

Unlike sonication, enzymatic digestion using micrococcal nuclease does not expose the sample to 

harsh denaturing conditions (high heat and detergent), so it gently fragments the chromatin while 

protecting the integrity of the protein-DNA complex. As shown below, enzyme-digested chromatin 

displays a more robust enrichment of target DNA loci than does sonicated chromatin, particularly 

with low frequency interactions. 

Titration identifies the optimal amount of antibody to use per IP. 


0.8 

0.7 

0.6 

0.5 

0.4 

0.3 

0.2 

0.1 

0 

1 µl 

Optimal Antibody Concentration 

2.5 µl 

5 µl 

10 µl 

p53 (1C12) Mouse mAb #2524 

20 µl 

40 µl 

Normal Rabbit 

IgG #2729 

SimpleChIP ® Enzymatic 

Chromatin IP Kit (Magnetic 

Beads) #9003: Chromatin IPs 

were performed with cross-linked 

chromatin from 4 x 10 6 HCT 116 cells 

treated with UV (100 J/m 2 followed 

by a 3 hr recovery) and the indicated 

amounts of p53 (1C12) Mouse mAb 

#2524 using #9003. The enriched 

DNA was quantified by real-time 

PCR using SimpleChIP ® Human 

CDKN1A Promoter Primers #6449, 

human MDM2 intron 2 primers, 

and SimpleChIP ® Human α Satellite 

Repeat Primers #4486. The amount 

of immunoprecipitated DNA in each 

sample is presented as a percent of 


CDKN1A 

MDM2 

Sat1α 

The right positive control is essential for assay reliability. 

Target DNA loci are 

immunoprecipitated 

better from enzymedigested 

chromatin 

than sonicated 

chromatin. 

SimpleChIP ® Plus Enzymatic 

Chromatin IP Kit (Magnetic Beads) 

#9005: Chromatin IPs were performed 

with enzyme-digested or sonicated 

chromatin and the indicated ChIPvalidated 

antibodies using #9005. 

The enriched DNA was quantified by 

real-time PCR using primers to the 

designated loci. The amount of immunoprecipitated 

DNA in each sample 

is presented as a percent of the total 

input chromatin. 

GAPDH RPL30 HoxA1 HoxA2 

SimpleChIP 

Sonicated 



35 

30 

25 

20 

15 

10 

5 

0 

6 

5 

4 

3 

2 

High Abundance, 

High Stability Interactions 

Histone H3 (D2B12) XP ® Rabbit 

mAb (ChIP Formulated) #4620 

Low Abundance, 

Low Stability Interactions 

Rpb1 CTD (4H8) 

Mouse mAb #2629 

Tri-Methyl-Histone H3 (Lys4) 

(C42D8) Rabbit mAb #9751 

Tri-Methyl-Histone H3 (Lys27) 

(C36B11) Rabbit mAb #9733 

A Histone H3 antibody will immunoprecipitate all genomic loci, whether transcriptionally active or 

inactive, so it functions as a universal control for tracking assay efficiency and reagent performance. 

Although Rpb1 antibody is often used as a positive control in other ChIP kits, it only binds active loci 

and therefore may not be suitable when examining transcriptionally inactive regions of the genome. 

H3 is a more reliable control than Rpb1. 


20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 


mAb (ChIP Formulated) #4620 

Rpb1 CTD (4H8) Mouse mAb #2629 Normal Rabbit IgG #2729 


mAb (ChIP Formulated) #4620: 

ChIP was performed with 10 μg 

of cross-linked chromatin and the 

indicated antibodies. The enriched 

DNA was quantified by qPCR. The 

amount of immunoprecipitated DNA 

in each sample is presented as a 

percent of the total input chromatin. 

γ-actin 

GAPDH 

RPL30 

HoxA1 

HoxA2 

MYT1 

1 

0 

Ezh2 (D2C9) XP ® 


SUZ12 (D39F6) XP ® 



Benefits of Enzyme-based ChIP 

To learn more about the major advantages of enzymatic digestion using micrococcal nuclease compared to sonication, 

please go to www.cellsignal.com/chipbenefits 


www.cellsignal.com/cstchip 

195



ChIP General Protocol 

Solutions and Reagents Included: 

Reagents Included in SimpleChIP ® and SimpleChIP ® Plus Enzymatic Chromatin IP Kits #9002, #9003, #9004, or #9005: 

Glycine Solution (10X), Buffer A (4X), Buffer B (4X), ChIP Buffer (10X), ChIP Elution Buffer (2X), 5 M NaCl, 0.5 M EDTA, 1 M DTT, 

DNA Binding Reagent A, DNA Wash Reagent B, DNA Elution Reagent C, DNA Spin Columns, Protease Inhibitor Cocktail (200X), 

RNAse A (10 mg/ml), Micrococcal Nuclease (2000 gel units/µl), Proteinase K (20 mg/ml), SimpleChIP ® Human RPL30 Exon 3 

Primers #7014, SimpleChIP ® Mouse RPL30 Intron 2 Primers #7015, Histone H3 (D2B12) XP ® Rabbit mAb (ChIP Formulated) 

#4620, Normal Rabbit IgG #2729, ChIP-Grade Protein G Magnetic Beads #9006 or ChIP-Grade Protein G Agarose Beads #9007 

Reagents Not Included: 

Formaldehyde (37%), Ethanol (96–100%), Isopropanol, 1X PBS (20X PBS #9808), Nuclease-free water, Taq DNA polymerase, 

dNTP Mix, For Kits #9002 and #9003 only: PMSF (#8553) (0.1 M stock), For Kits #9003 and #9005 only: 6-Tube Magnetic 

Separation Rack #7017 

I. Tissue Cross-linking and Sample Preparation 

IMPORTANT: Section I exclusive to use of SimpleChIP ® Plus Kits (#9004 & #9005) 

When harvesting tissue, remove unwanted material such as fat and necrotic material from the sample. Tissue can then be 

processed and cross-linked immediately, or frozen on dry ice for processing later. For optimal chromatin yield and ChIP results, 

use 25 mg of tissue for each IP to be performed. The chromatin yield does vary between tissue types and some tissues may 

require more than 25 mg for each IP. Please see Troubleshooting Guide, Section A for more information regarding the expected 

chromatin yield for different types of tissue. One additional chromatin sample should be processed for Analysis of Chromatin 

Digestion and Concentration (Section IV). 

Before starting: 

• Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X glycine solution. Make sure PIC is completely thawed. 

• Prepare 3 ml of 1X Phosphate Buffered Saline (PBS) + 15 μl 200X PIC per 25 mg of tissue to be processed and place on ice. 

• Prepare 45 μl of 37% formaldehyde per 25 mg of tissue to be processed and keep at room temperature. Use fresh formaldehyde 

that is not past the manufacturer’s expiration date. 

A. Cross-linking 

1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be performed. 

2. Place tissue sample in a 60 mm or 100 mm dish and finely mince using a clean scalpel or razor blade. Keep dish on ice. It is 

important to keep the tissue cold to avoid protein degradation. 

3. Transfer minced tissue to a 15 ml conical tube. 

4. Add 1 ml of 1X PBS + PIC per 25 mg tissue to the conical tube. 

5. To crosslink proteins to DNA, add 45 μl of 37% formaldehyde per 1 ml of 1X PBS + PIC and rock at room temp for 20 min. 

Final formaldehyde concentration is 1.5%. 

6. Stop cross-linking by adding 100 μl of 10x glycine per 1 ml of 1X PBS + PIC and mix for 5 min at room temperature. 

7. Centrifuge tissue at 1,500 rpm in a bench top centrifuge for 5 min at 4°C. 

8. Remove supernatant and wash with 1 ml 1X PBS + PIC per 25 mg tissue. 

9. Repeat centrifugation (Section A, Step 7). 

10. Remove supernatant and resuspend tissue in 1 ml 1X PBS + PIC per 25 mg tissue and store on ice. Disaggregate tissue into 

single-cell suspension using a Medimachine (Section B) or Dounce homogenizer (Section C). 

B. Tissue Disaggregation Using Medimachine from BD Biosciences (part #340587) 

1. Cut off the end of a 1 ml pipette tip to enlarge the opening for transfer of tissue chunks. 

2. Transfer 1 ml of tissue resuspended in 1X PBS + PIC into the top chamber of a medicon filter (50 µm mesh; BD Part #340592). 

3. Grind tissue for 2 min according to manufacturer’s instructions. 

4. Collect cell suspension from the bottom chamber of the medicon using a 1 ml syringe and 18-gauge blunt needle. 

Transfer cell suspension to a 15 ml conical tube and place on ice. 

5. Repeat steps 2–4 until all the tissue is processed into a homogenous suspension. 

6. If more grinding is necessary, add more 1X PBS + PIC to tissue. Repeat steps 2–5 until all tissue is ground into a 

homogeneous suspension. 

7. Check for single-cell suspension by microscope (optional). 

8. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C. 

9. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III). 

C. Tissue Disaggregation Using a Dounce Homogenizer 

1. Transfer tissue resuspended in 1X PBS + PIC to a Dounce homogenizer. 

2. Disaggregate tissue pieces with 20–25 strokes. Check for single-cell suspension by microscope (optional). 

3. Transfer cell suspension to a 15 ml conical tube and centrifuge at 1,500 rpm in a bench top centrifuge for 5 min at 4°C. 

4. Remove supernatant from cells and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III). 

II. Cell Culture Cross-linking and Sample Preparation 

For optimal ChIP results, use approximately 4 x 10 6 cells for each IP to be performed. For HeLa cells, this is equivalent to half 

of a 15 cm culture dish containing cells that are 90% confluent in 20 ml of growth medium. One additional sample should be 

processed for Analysis of Chromatin Digestion and Concentration (Section IV). Include one extra dish of cells in experiment to 

be used for determination of cell number using a hemocytometer. 



• Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine Solution. Make sure PIC is completely thawed. 

• Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 μl 200X PIC per 15 cm dish to be processed and place on ice. 

• Prepare 40 ml of 1X PBS per 15 cm dish to be processed and place on ice. 

• Prepare 540 μl of 37% formaldehyde per 15 cm dish of cells to be processed and keep at room temperature. Use fresh 

formaldehyde that is not past the manufacturer’s expiration date. 

1. To crosslink proteins to DNA, add 540 μl of 37% formaldehyde to each 15 cm culture dish containing 20 ml medium. Swirl 

briefly to mix and incubate 10 min at room temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde 

may result in a color change of the medium. 

2. Add 2 ml of 10X glycine solution to each 15 cm dish containing 20 ml medium, swirl briefly to mix, and incubate 5 min at 

room temperature. Addition of glycine may result in a color change of the medium. 

3. For suspension cells, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm in a bench top centrifuge for 5 min 

at 4°C and wash pellet two times with 20 ml ice-cold 1X PBS. Remove supernatant and immediately continue with Nuclei 

Preparation and Chromatin Digestion (Section III). 

4. For adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X PBS, completely removing wash from 

culture dish each time. 

5. Add 2 ml ice-cold 1X PBS + PIC to each 15 cm dish. Scrape cells into cold buffer. Combine cells from all culture dishes into 

one 15 ml conical tube. 

6. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4°C. Remove supernatant and immediately continue 

with Nuclei Preparation and Chromatin Digestion (Section III). 

III. Nuclei Preparation and Chromatin Digestion 

One IP preparation is defined as 25 mg of disaggregated tissue or 4 x 10 6 tissue culture cells. 


• Remove and warm 200X Protease Inhibitor Cocktail (PIC) and 1 M DTT. Make sure both are completely thawed and DTT 

crystals are completely in solution. 

• Remove and warm 10X ChIP buffer and ensure SDS is completely in solution. 

• Prepare 1 ml 1X buffer A (250 μl 4X buffer A + 750 μl water) + 0.5 μl 1 M DTT + 5 μl 200X PIC per IP prep and place on ice. 

• Prepare 1.1 ml 1X Buffer B (275 μl 4X buffer B + 825 μl water) + 0.55 μl 1 M DTT per IP prep and place on ice. 

• Prepare 100 μl 1X ChIP buffer (10 μl 10X ChIP Buffer + 90 μl water) + 0.5 μl 200X PIC per IP prep and place on ice. 

1. Resuspend cells in 1 ml ice-cold buffer A + DTT + PIC per IP prep. Incubate on ice for 10 min. Mix by inverting tube every 

3 min. 

2. Pellet nuclei by centrifugation at 3,000 rpm in a bench top centrifuge for 5 min at 4°C. Remove supernatant and resuspend 

pellet in 1 ml ice-cold buffer B + DTT per IP prep. Repeat centrifugation, remove supernatant, and resuspend pellet in 100 μl 

Buffer B + DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per tube. 

3. Add 0.5 μl of micrococcal nuclease per IP prep, mix by inverting tube several times and incubate for 20 min at 37°C with 

frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3–5 min. The amount of micrococcal 

nuclease required to digest DNA to the optimal length may need to be determined empirically for individual tissues and 

cell lines (see Troubleshooting Guide, Section B). HeLa nuclei digested with 0.5 μl micrococcal nuclease per 4 x 10 6 cells and 

mouse liver tissue digested with 0.5 μl micrococcal nuclease per 25 mg of tissue gave the appropriate length DNA fragments. 

4. Stop digest by adding 10 μl of 0.5 M EDTA per IP prep and placing tube on ice. 

5. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant. 

6. Resuspend nuclear pellet in 100 μl of 1X ChIP buffer + PIC per IP prep and incubate on ice for 10 min. 

7. Sonicate up to 500 μl of lysate per 1.5 ml microcentrifuge tube with several pulses to break nuclear membrane. Incubate 

samples for 30 sec on wet ice between pulses. Optimal conditions required for complete lysis of nuclei can be determined by 

observing nuclei on a light microscope before and after sonication. 

HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator 

at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a Dounce 

homogenizer; however, lysis may not be as complete. 

8. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C. 

9. Transfer supernatant to a new tube. This is the cross-linked chromatin preparation, which should be stored at -80°C until 

further use. Remove 50 μl of the chromatin preparation for Analysis of Chromatin Digestion and Concentration (Section IV). 

IV. Analysis of Chromatin Digestion and Concentration (Recommended Step) 

1. To the 50 μl chromatin sample (Step 9 in Section III), add 100 μl nuclease-free water, 6 μl 5 M NaCl, and 2 μl RNAse A. Vortex 

to mix and incubate samples at 37°C for 30 min. 

2. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate samples at 65°C for 2 hr. 

3. Purify DNA from samples using DNA purification spin columns as described in Section VII. 

4. After purification of DNA, remove a 10 μl sample and determine DNA fragment size by electrophoresis on a 1% agarose gel 

with a 100 bp DNA marker. DNA should be digested to a length of approximately 150–900 bp (1–6 nucleosomes). 

5. To determine DNA concentration, transfer 2 μl of purified DNA to 98 μl nuclease-free water to give a 50-fold dilution and 

read the OD260. The concentration of DNA in μg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 

200 μg/ml. 

NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion 

of chromatin may diminish signal in the PCR quantification. Under-digestion of chromatin may lead to increased background 

signal and lower resolution. Adding too little chromatin to the IP may result in diminished signal in the PCR quantification. A 

protocol for optimization of chromatin digestion can be found in the Troubleshooting Guide. 

www.cellsignal.com/cstprotocols 197



ChIP Companion 

Products 

We offer a comprehensive list of 

companion products that are validated 

in-house with our protocols so that you 

can get the reliable reagents you need 

to complete your experiment. 

www.cellsignal.com/chipcompanions 

V. Chromatin IP 

For optimal ChIP results, use approximately 5–10 μg of digested, cross-linked chromatin (as determined in Section IV) per IP. 

This should be roughly equivalent to a single 100 μl IP prep from 25 mg of disaggregated tissue or 4 x 10 6 tissue culture cells. 

Typically, 100 μl of digested chromatin is diluted into 400 μl 1X ChIP Buffer prior to the addition of antibodies. However, if more 

than 100 μl of chromatin is required per IP, the cross-linked chromatin preparation does not need to be diluted as described 

below. Antibodies can be added directly to the undiluted chromatin preparation for IP of chromatin complexes. 


• Remove and warm 200X Protease Inhibitor Cocktail (PIC). Make sure PIC is completely thawed. 

• Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution. 

• Thaw digested chromatin preparation (Section III, Step 9) and place on ice. 

• Prepare low salt wash: 3 ml 1X ChIP buffer (300 μl 10X ChIP buffer + 2.7 ml H 2 0) per IP prep. Store at room temperature 

until use. 

• Prepare high salt wash: 1 ml 1X ChIP buffer (100 μl 10X ChIP buffer + 900 μl H 2 0) + 70 μl 5M NaCl per IP. Store at room 

temperature until use. 

1. In one tube, prepare enough 1X ChIP buffer for the dilution of digested chromatin into the desired number of IPs: 400 μl 

of 1X ChIP buffer (40 μl of 10X ChIP buffer + 360 μl H 2 0) + 2 μl 200X PIC per IP. When determining the number of IPs, remember 

to include the positive control Histone H3 (D2B12) XP ® Rabbit mAb and negative control Normal Rabbit IgG antibody 

samples. Place mix on ice. 

2. To the prepared 1X ChIP buffer, add the equivalent of 100 μl (5–10 μg of chromatin) of the digested, cross-linked chromatin 

preparation (Section III, Step 9) per IP. For example, for 10 IPs, prepare a tube containing 4 ml 1X ChIP Buffer (400 μl 10X 

ChIP buffer + 3.6 ml water) + 20 μl 200X PIC + 1 ml digested chromatin preparation. 

3. Remove a 10 μl sample of the diluted chromatin and transfer to a microfuge tube. This is your 2% input sample, which can 

be stored at -20°C until further use (Step 1 in Section VI). 

4. For each IP, transfer 500 μl of the diluted chromatin to a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. 

The amount of antibody required per IP varies and should be determined by the user. For the positive control Histone 

H3 (D2B12) XP ® Rabbit mAb add 10 μl to the IP sample. For the negative control, Normal Rabbit IgG, add 1 μl (1 μg) to 2 μl 

(2 μg) to the IP sample. Incubate IP samples 4 hr to overnight at 4°C with rotation. 

5. a. Resuspend ChIP-Grade Protein G Agarose Beads by gently vortexing. Immediately add 30 μl of Protein G Agarose Beads to 

each IP reaction and incubate for 2 hr at 4°C with rotation. 

b. Resuspend ChIP-Grade Protein G Magnetic Beads by gently vortexing. Immediately add 30 μl of Protein G Magnetic Beads 

to each IP reaction and incubate for 2 hr at 4°C with rotation. 

6. a. Pellet Protein G Agarose Beads in each IP by brief 1 min centrifugation at 6,000 rpm in a microcentrifuge and remove 

supernatant. 

b. Pellet Protein G Magnetic Beads in each IP by placing the tubes in a Magnetic Separation Rack. Wait 1–2 min for solution 

to clear and then carefully remove supernatant. 

7. Wash Protein G Beads by adding 1 ml of low salt wash to the beads and incubate at 4°C for 5 min with rotation. Repeat 

Steps 6 and 7 two additional times for a total of 3 low salt washes. 

8. Add 1 ml of high salt wash to the beads and incubate at 4°C for 5 min with rotation. 

9. a. Pellet Protein G Agarose Beads in each IP by brief 1 min centrifugation at 6,000 rpm in a microcentrifuge. Remove 

supernatant and immediately proceed to Section VI. 

b. Pellet Protein G Magnetic Beads in each IP by placing the tubes in a Magnetic Separation Rack. Wait 1–2 min for solution to 

clear and then carefully remove supernatant and proceed to Section VI. 

VI. Elution of Chromatin from Antibody/Protein G Agarose Beads and Reversal of Cross-links 


• Remove and warm 2X ChIP elution buffer in a 37°C water bath and ensure SDS is in solution. 

• Set a water bath or thermomixer to 65°C. 

• Prepare 150 μl 1X ChIP elution buffer (75 μl 2X ChIP elution buffer + 75 μl water) for each IP and the 2% input sample. 

1. Add 150 μl of the 1X ChIP elution buffer to the 2% input sample tube and set aside at room temperature until Step 6. 

2. Add 150 μl 1X ChIP elution buffer to each IP sample. 

3. Elute chromatin from the antibody/Protein G Beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works 

best for this step. Alternatively, elutions can be performed at room temperature with rotation, but may not be as complete. 

4. a. Pellet Protein G Agarose Beads by brief 1 min centrifugation at 6,000 rpm in a microcentrifuge. 

b. Pellet Protein G Magnetic Beads by placing the tubes in a Magnetic Separation Rack and wait 1–2 min for solution to clear. 

5. Carefully transfer eluted chromatin supernatant to a new tube. 

6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 μl 5 M NaCl and 2 μl Proteinase K, 

and incubate 2 hr at 65°C. This incubation can be extended overnight. 

7. Immediately proceed to Section VII. Alternatively, samples can be stored at -20°C. However, to avoid formation of a precipitate, 

be sure to warm samples to room temperature before adding DNA binding reagent A (Section VII, Step 1). 

VII. DNA Purification Using Spin Columns 


• Add 12 ml of isopropanol to DNA binding reagent A and 24 ml of ethanol (96–100%) to DNA wash reagent B before use. 

These steps only have to be performed once prior to the first set of DNA purifications. 

• Remove one DNA purification spin column and collection tube for each DNA sample (Section VI). 

1. Add 600 μl DNA binding reagent A to each DNA sample and vortex briefly. 

4 volumes of DNA binding reagent A should be used for every 1 volume of sample. 

2. Transfer 375 μl of each sample from Step 1 to a DNA purification spin column in collection tube. 

3. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec. 

4. Remove spin column from the collection tube and discard the liquid. Replace spin column in the collection tube. 

5. Transfer remaining 375 μl of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4. 

6. Add 700 μl of DNA wash reagent B to the spin column in collection tube. 


8. Remove spin column from the collection tube and discard the liquid. Replace spin column in the collection tube. 


10. Discard collection tube and liquid. Retain spin column. 

11. Add 50 μl of DNA elution reagent C to each spin column and place into a clean 1.5 ml microcentrifuge tube. 

12. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec to elute DNA. 

13. Remove and discard DNA purification spin column. Eluate is now purified DNA. Samples can be stored at -20°C. 

VIII. Quantification of DNA by PCR 

Recommendations: 

• Use Filter-tip pipette tips to minimize risk of contamination. 

• The control primers included in the kit are specific for the human or mouse RPL30 gene and can be used for either standard 

PCR or quantitative real-time PCR. If the user is performing ChIPs from another species, it is recommended that the user 

design the appropriate specific primers to DNA and determine the optimal PCR conditions. 

• A Hot-Start Taq polymerase is recommended to minimize the risk of non-specific PCR products. 

• PCR primer selection is critical. Primers should be designed with close adherence to the following criteria: 

Primer length: 24 nucleotides 

Optimum Tm: 60°C 

Optimum GC: 50% 

Amplicon size: 150–200 bp (for standard PCR) 80–160 bp (for real-time quantitative PCR) 

Standard PCR Method: 

1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 2% 

input sample, the positive control histone H3 sample, the negative control normal rabbit IgG sample, and a tube with no DNA 

to control for DNA contamination. 

2. Add 2 μl of the appropriate DNA sample to each tube. 

3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for 

loss of volume. Add 18 μl of master mix to each reaction tube. 

Reagent: Volume for 1 PCR Reaction (18 µl), Nuclease-free H 2 O: 12.5 µl, 10X PCR Buffer: 2.0 µl, 4 mM dNTP Mix: 1.0 µl, 

5 µM RPL30 Primers: 2.0 µl, Taq DNA Polymerase: 0.5 µl 

4. Start the following PCR reaction program: 

a. Initial denaturation 95°C 5 min 

b. Denature 95°C 30 sec 

c. Anneal 62°C 30 sec 

d. Extension 72°C 30 sec 

e. Repeat Steps b–d for a total of 34 cycles 

f. Final Extension 72°C 5 min 

5. Remove 10 μl of each PCR product for analysis by 2% agarose gel or 10% poly-acrylamide gel electrophoresis with a 100 bp 

DNA marker. The expected size of the PCR product is 161 bp for human RPL30 and 159 bp for mouse RPL30. 

Real-Time Quantitative PCR Method: 

1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. PCR 

reactions should include the positive control histone H3 sample, the negative control normal rabbit IgG sample, a tube with no 

DNA to control for contamination, and a serial dilution of the 2% input chromatin DNA (undiluted, 1:5, 1:25, 1:125) to create 

a standard curve and determine the efficiency of amplification. 

2. Add 2 μl of the appropriate DNA sample to each tube or well of the PCR plate. 

3. Prepare a master reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. 

Add 18 μl of reaction mix to each PCR reaction tube or well. 

Reagent: Volume for 1 PCR Reaction (18 µl), Nuclease-free H 2 O: 6 µl 5 µM RPL30 Primers: 2 µl, 2X SYBR ® -Green Reaction 

Mix: 10 µl 

4. Start the following PCR reaction program: 

a. Initial denaturation 95°C, 3 min 

b. Denature 95°C, 15 sec 

c. Anneal and extension: 60°C, 60 sec 

d. Repeat steps b and c for a total of 40 cycles. 

5. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate 

the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained 

from each IP are expressed as a percent of the total input chromatin. 

Percent Input = 2% x 2 (C[T] 2% Input Sample – C[T] IP Sample) C[T] = C T = Threshold cycle of PCR reaction 


www.cellsignal.com/cstprotocols 

199



ChIP Troubleshooting Guide 

A. Expected Chromatin Yield 

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin can vary significantly between tissue types. 

The table below provides a range for the expected yield of chromatin from 25 mg of tissue compared to 4 x 10 6 HeLa cells, and 

the expected DNA concentration, as determined in Section IV of the protocol. For each tissue type, disaggregation using a BD 

Medimachine system (BD Biosciences) or a Dounce homogenizer yielded similar amounts of chromatin. However, chromatin 

processed from tissues disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin processed 

from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of 

brain tissue, as the Medimachine does not adequately disaggregate brain tissue into a single-cell suspension. For optimal ChIP 

results, we recommend using 5–10 µg of digested, cross-linked chromatin per IP; therefore, some tissues may require harvesting 

more than 25 mg per each IP. 

Total Chromatin Yield 

Expected DNA Concentration 

Spleen 

20–30 µg per 25 mg tissue 200–300 µg/ml 

Liver 


Kidney 


Total Chromatin Yield 

Expected DNA Concentration 

Brain 


Heart 


HeLa 

10–15 µg per 4 x 10 6 cells 100–150 µg/ml 

B. Optimization of Chromatin Digestion 

Optimal conditions for the digestion of cross-linked chromatin DNA to 150–900 bp in length is highly dependent on the ratio of 

micrococcal nuclease to the amount of tissue or number of cells used in the digest. Below is a protocol for determination of the 

optimal digestion conditions for a specific tissue or cell type. 

1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 10 7 cells (equivalent of 5 IP preps), as described in Sections I, II, 

and III. Stop after Step 2 of Section III and proceed as described below. 

2. Transfer 100 μl of the nuclei preparation into 5 individual 1.5 ml microcentrifuge tubes and place on ice. 

3. Add 3 μl micrococcal nuclease stock to 27 μl of 1X Buffer B + DTT (1:10 dilution of enzyme). 

4. To each of the 5 tubes in Step 2, add 0 μl, 2.5 μl, 5 μl, 7.5 μl, or 10 μl of the diluted micrococcal nuclease, mix by inverting 

tube several times and incubate for 20 min at 37°C with frequent mixing. 

5. Stop each digest by adding 10 μl of 0.5 M EDTA and placing tubes on ice. 

6. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4°C and remove supernatant. 

7. Resuspend nuclear pellet in 200 μl of 1X ChIP buffer + PIC. Incubate on ice for 10 min. 

8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses. 

Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei on a light microscope before 

and after sonication. HeLa nuclei were completely lysed after 3 sets of 20 sec pulses using a VirTis Virsonic 100 Ultrasonic 

Homogenizer/Sonicator set at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be lysed by homogenizing the lysate 

20 times in a Dounce homogenizer; however, lysis may not be as complete. 

9. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4°C. 

10. Transfer 50 μl of each of the sonicated lysates to new microfuge tubes. 

11. To each 50 μl sample, add 100 μl nuclease-free water, 6 μl 5 M NaCl and 2 μl RNAse A. Vortex to mix and incubate 

samples at 37°C for 30 min. 

12. To each RNAse A-digested sample, add 2 μl Proteinase K. Vortex to mix and incubate sample at 65°C for 2 hr. 

13. Remove 20 μl of each sample and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp 

DNA marker. 

14. Observe which of the digestion conditions produces DNA in the desired range of 150–900 base pairs (1–6 nucleosomes). 

The volume of diluted micrococcal nuclease that produces the desired size of DNA fragments using this optimization protocol 

is equivalent to 10 times the volume of micrococcal nuclease stock that should be added to one IP preparation (25 mg of 

disaggregated tissue cells or 4 X 10 6 tissue culture cells) to produce the desired size of DNA fragments. For example, if 5 μl 

of diluted micrococcal nuclease produces DNA fragments of 150–900 bp in this protocol, then 0.5 μl of stock micrococcal 

nuclease should be added to one IP preparation during the digestion of chromatin (Section III). 

15. If results indicate that DNA is not in the desired size range, then repeat optimization protocol, adjusting the amount of 

micrococcal nuclease in each digest accordingly. Alternatively, the digestion time can be changed to increase or decrease 

the extent of DNA fragmentation. 

Problem Possible Causes Recommendation 

Concentration of the 

digested chromatin is too 

low (low chromatin yield). 

Chromatin is under-digested 

and fragments are too large 

(greater than 900 bp). Large 

chromatin fragments can lead 

to increased background and 

lower resolution. 

Chromatin is over-digested 

and fragments are too small 

(exclusively 150 bp mononucleosome 

length). Complete 

digestion of chromatin to 

mono-nucleosome length DNA 

may diminish signal during PCR 

quantification, especially for 

amplicons greater than 150 bp 

in length. 

No product or very little product 

in the input PCR reactions. 

No product in the positive 

control histone H3-IP RPL30 

PCR reaction. 

Quantity of product in the 

negative control Rabbit IgG-IP 

and positive control histone 

H3-IP PCR reactions is equivalent 

(high background signal). 

No product in the Experimental 

Antibody-IP PCR reaction. 

Not enough tissue or cells were 

added to the chromatin digestion or 

cell nuclei were not completely lysed 

after digestion. 

Too many cells or not enough 

micrococcal nuclease was added to 

the chromatin digestion. 

Tissue or cells may have been over 

cross-linked. Cross-linking for longer 

than 10 min may inhibit digestion of 

chromatin. 

Not enough cells or too much 

micrococcal nuclease added to the 

chromatin digestion. 

Not enough DNA added to the PCR 

reaction or conditions are not optimal. 

PCR amplified region may span 

nucleosome-free region. 

Not enough chromatin added to the 

IP or chromatin is over-digested. 

Not enough chromatin or antibody 

added to the IP reaction or IP incubation 

time is too short. 

Incomplete elution of chromatin from 

Protein G beads. 

Too much or not enough chromatin 

added to the IP reaction. Alternatively, 

too much antibody added to 

the IP reaction. 

Too much DNA added to the PCR 

reaction or too many cycles of 

amplification. 

Not enough DNA added to the PCR 

reaction. 

Not enough antibody added to the 

IP reaction. 

Antibody does not work for ChIP. 

Add additional chromatin to each IP to give at least 

5 μg/IP and continue with protocol. 

Weigh tissue or count a separate plate of cells prior to 

cross-linking to determine accurate cell number. Some 

tissues may require processing of more than 25 mg per 

IP. The amount of tissue can be increased to 50 mg per 

IP, while still maintaining efficient chromatin fragmentation 

and extraction. 

Increase the number of sonications following chromatin 

digestion. Visualize cell nuclei under microscope before 

and after sonication to confirm complete lysis of nuclei. 


cross-linking to determine accurate cell number. Add 

less tissue or cells, or more micrococcal nuclease to 

the chromatin digest. See Section B for optimization of 

chromatin digestion. 

Perform a time course at a fixed formaldehyde concentration. 

Shorten the time of cross-linking 

to 10 min or less. 


cross-linking to determine accurate cell number. Add 

more tissue or cells, or less micrococcal nuclease to 

the chromatin digest. See Section B of troubleshooting 

guide for optimization of chromatin digestion. 

Add more DNA to the PCR reaction or increase the 

number of amplification cycles. 

Optimize the PCR conditions for experimental primer 

set using purified DNA from cross-linked and digested 

chromatin. Design a different primer set and decrease 

length of amplicon to less than 150 bp (see primer 

design recommendations in Protocol Section VIII). 

For optimal ChIP results, add 5–10 μg chromatin per IP. 

Be sure to add 5–10 μg of chromatin and 10 μl 

of antibody to each IP reaction and incubate with 

antibody overnight and an additional 2 hr after adding 

Protein G beads. 

Elution of chromatin from Protein G beads is optimal at 

65°C with frequent mixing to keep beads suspended 

in solution. 

For optimal ChIP results, add 5–10 µg of chromatin 

and 10 μl of histone H3 antibody to each IP reaction. 

Reduce the amount of normal rabbit IgG to 1 μl per IP. 

Add less DNA to the PCR reaction or decrease the 

number of PCR cycles. It is very important that 

the PCR products are analyzed within the linear 

amplification phase of PCR. Otherwise, the differences 

in quantities of starting DNA cannot be accurately 

measured. Alternatively, quantify immunoprecipitations 

using real-time quantitative PCR. 

Add more DNA to the PCR reaction or increase the 

number of amplification cycles. 

Typically a range of 1–5 μg of antibody are added to 

the IP reaction; however, the exact amount depends 

greatly on the individual antibody. Increase the amount 

of antibody added to the IP. 

Find an alternate antibody source. 


Guide to Successful ChIP 

Helpful tips and explanations to support your ChIP experiments are available in “A Guide to Successful Chromatin IP”. 

Request a copy at www.cellsignal.com/chipsuccess 


201

Events 

10 


chapter 

Phosphorylation 

of intracellular 

Stat5 (Tyr694) 

is stimulated by 

GM-CSF treatment. 


Phospho-Stat5 (Tyr694) (D47E7) XP ® 

Rabbit mAb #4322: Flow cytometric 

analysis of TF-1 cells, untreated (blue) 

or treated with hGM-CSF #8922 (green), 

using #4322. 

Btk (D3H5) Rabbit mAb #8547: 

Human whole blood was fixed and 

permeabilized as outlined in the CST 

Flow Alternate Protocol and stained 

using #8547. Forward scatter (FSC) 

and side scatter (SSC) were used to 

gate on lymphocyte cells (A). Samples 

were co-stained using CD3-PE and 

CD19-APC to distinguish T and B 

cell subpopulations (B). B (red) and 

T (blue) cell population gates were 

applied to a histogram depicting the 

mean fluorescence intensity of Btk (C). 

Flow Cytometry (F) 

Flow cytometry is a well-established technology allowing phenotypic, molecular, and functional characterization 

of single cells in suspension. The unique multi-parameter capability of this platform enables 

the simultaneous detection and quantification of multiple intracellular and extracellular targets in the 

same sample. Flow cytometry is highly sensitive and can be used to detect cellular proteins within 

distinct sub populations from heterogeneous samples. Traditionally used for the phenotypic analysis of 

normal and malignant blood cells, flow cytometry is now also routinely used to study complex intracellular 

signaling. 

Intracellular flow cytometry can be used to analyze signaling changes in response to stimuli within 

specific cell types and subpopulations. Cells are fixed and permeabilized to allow antibody penetration 

and subsequent binding to targets without disrupting cellular morphology. Several fixation and permeabilization 

protocols have been developed for target-specific antibodies to enable intracellular staining 

alone or in combination with surface markers, and to maximize signal-to-noise. 

There are numerous methodologies available to perform intracellular flow cytometry. Below we discuss 

several key processes that will help achieve experimental success, particularly when studying posttranslational 

modifications (PTMs). 

Flow Cytometry Tips for Success 

Appropriate fixation and permeabilization methods are 

crucial for successful detection of intracellular targets. 

Flow cytometric analysis of PTMs, such as phosphorylation, requires fixation of cells within a critical 

window of pathway activation. Fixation using 4% formaldehyde (methanol-free) optimally crosslinks 

intracellular proteins and any associated modifications. Furthermore, enzymatic activity responsible for 

removing modifications such as phosphatases can be inactivated. A lower concentration of formaldehyde 

may be necessary for some targets if the extent of crosslinking masks the target epitope. 

Permeabilization with ice-cold 90% methanol added dropwise maintains structural cell integrity while 

allowing efficient antibody penetration without denaturing PTM epitopes that have previously been 

aldehyde fixed (1). 

Protocol alterations enable simultaneous detection of 

intracellular and extracellular targets by flow cytometry. 

Ex vivo stimulation of whole blood allows for perturbation of peripheral blood cells within the blood 

matrix, resulting in a more physiologically relevant model of pathway signaling and other cellular 

processes. Fixation in 4% formaldehyde stabilizes protein interactions and subsequent treatment with 

0.1% Trition X-100 lyses red blood cells (RBCs) (2). This allows permeabilization with 50% methanol 

to preserve surface marker integrity, enabling the simultaneous analysis of both surface and intracellular 

targets. 

Btk, an intracellular tyrosine kinase, is selectively expressed in B cells. 

SSC-H 

1000 

800 

600 

400 

200 

A 10 B 4 

100 

C 

0 200 400 600 800 1000 

FSC-H 


CD3-PE 

10 3 

10 2 

10 1 

T cells 

B cells 

Events 

10 0 

10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 

CD19-APC 

Btk 

80 

60 

40 

20 

B cells 

T cells 

Consider the impact of basal modification levels 

when assessing signal transduction events. 

It is always important to account for the complexity of cellular systems when designing an experiment. 

In some instances, basal modification levels may be high and therefore, stimulation of the pathway of 

interest does not provide any additional detectable signal, which can be interpreted as a false negative 

result. This event is common with phosphorylated proteins. To address this issue, the fixed and permeabilized 

sample can be treated with phosphatase to remove phosphate groups from proteins and then 

examined for decreased signal that indicates high basal phosphorylation levels. Endogenous phosphorylation 

levels in live cells can be brought down by serum starvation or by kinase inhibitor treatment. Conversely, 

transient phosphorylation events may be difficult to capture due to endogenous phosphatase 

activity, in which case treatment with a phosphatase inhibitor can help preserve phosphorylation signal. 

Phosphatase treatment reveals basal 

phosphorylation levels of S6 ribosomal protein. 

Side Scatter 

Intracellular flow cytometry can be used 

to detect and quantify cytoplasmic cytokines. 

Adding Brefeldin A to a cell sample during stimulation prevents secretion of cytokines by blocking intracellular 

vesicle trafficking. Thus, cytokines accumulate in the cells, allowing for their detection. In the 

example shown, CST’s standard flow cytometry protocol was used to immunostain cell surface markers 

in conjunction with an intracellular cytokine Interferon-γ. With this protocol, further multiplexing using 

antibodies against phosphorylated proteins would allow the simultaneous analysis of phenotype, 

protein activation and functional endpoint, on a single cell basis, within a single sample. 

Cytoplasmic Interferon-γ can be monitored with intracellular flow cytometry. 

IFN-γ (D3H2) XP ® Rabbit mAb #8455: 

Flow cytometric analysis of human 

peripheral blood cells, untreated (left) or 

treated (right) with TPA #4174 (40 nM, 

4 hr), Ionomycin #9995 (2 μM, 4 hr), 

and Brefeldin A #9972 (1 μg/ml, last 3 

hr of stimulation), using a CD3 antibody 

and #8455. Analysis was performed on 

cells in the lymphocyte gate. The CD3 

antibody (Y axis) was used to identify T 

cells. Note the induction of IFN-γ (X axis) 

in treated, CD3+ cells (right). 

A B C 

CD3 

Phospho-S6 Ribosomal Protein (Ser235/236) 

10 4 

10 3 

10 2 

10 1 

10 0 

10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 

IFN-γ 

References: 

1. Krutzik, P.O. and Nolan, G.P. (2003) Cytometry A. 55, 61–70. 

2. Chow, S., Hedley, D., Grom, P., Magari, R., Jacobberger, J.W., and Shankey, T.V. (2005) Cytometry A. 67, 4–17. 

10: Flow Cytometry (F) 

Phospho-S6 Ribosomal Protein 

(Ser235/236) (D57.2.2E) XP ® Rabbit 


#4803: Human whole blood was fixed 

and permeabilized as outlined in the CST 

Flow Alternate Protocol, and untreated 

(A), treated with λ phosphatase (B), or 

treated with TPA #4174 (C), before being 

stained using #4803. 

Alexa Fluor® 

Conjugated 

Antibodies 

For the most up-to-date list of Alexa 

Fluor ® conjugates that are optimized 

for flow cytometry, please go to 

www.cellsignal.com/alexafluor 

www.cellsignal.com/cstflow 

203


chapter 10: Flow Cytometry (F) 

PE conjugated 

antibodies deliver 

robust Stoke’s 

shifts and are ideal 

for multiplexing. 

Events 

Phospho-Akt (Ser473) 

Anti-rabbit IgG (H+L), F(ab’) 2 

Fragment (PE Conjugate) #8885: 

Flow cytometric analysis of Jurkat 

cells, untreated (green) or treated 

with LY294002 #9901, Wortmannin 

#9951, and U0126 #9903 (blue), using 

Phospho-Akt (Ser473) (D9E) XP ® Rabbit 

mAb #4060 detected with #8885. 

Flow Cytometry General Protocol 

A. Solutions and Reagents 

NOTE: Prepare solutions with reverse osmosis deionized or equivalent grade water (dH 2 0). 

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2 O, mix. 

2. 16% Formaldehyde (methanol free) 

3. 100% Methanol 

4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C. 

5. Secondary Antibodies: Anti-mouse (#4408, #8890, #4410, #8887), Anti-rabbit (#4412, #8889, #4414, #8885), Anti-rat 

(#4416, #4418) 

B. Fixation 

1. Collect cells by centrifugation and aspirate supernatant. 

2. Resuspend cells in 0.5–1 ml 1X PBS. Add appropriate volume of formaldehyde to obtain a final concentration of 

4% (e.g. 750 µl 1X PBS + 250 µl 16% formaldehyde). 

3. Fix for 10 min in a 37°C water bath. 

4. Chill tubes on ice for 1 min. 

5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) 

or store cells in 1X PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C). 

C. Permeabilization 

NOTE: This step is critical for many CST antibodies. 

1. Add ice-cold 100% methanol drop-wise to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. 

Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above. 

2. Permeabilize for a minimum of 10 min on ice. 

3. Proceed with Immunostaining (Section D) or store cells at -20°C in 90% methanol. 

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining 

1. Aliquot 100 μl fresh whole blood per assay tube. 

2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc. 

3. Add 65 μl of 10% formaldehyde to each tube. 

4. Vortex briefly and let stand for 15 min at room temperature. 

5. Add 1 ml of 0.1% Triton X-100 to each tube. 

6. Vortex and let stand for 30 min at room temperature. 

7. Add 1 ml incubation buffer. 

8. Pellet cells by centrifugation and aspirate supernatant. 

9. Repeat Steps 7 and 8. 

10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use). 

11. Incubate at least 10 min on ice. 

12. Proceed with staining or store cells at -20°C in 50% methanol. 

C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies 

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. 

1. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat. 

2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer. 

3. Incubate for 30–60 min at room temperature. 

4. Wash by centrifugation in 2–3 ml incubation buffer. 

5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in incubation buffer according to the manufacturer’s 

recommendations. 

6. Incubate for 30 min at room temperature. 

7. Wash by centrifugation in 2–3 ml incubation buffer. 

8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer. 

Cyclin B1 

Protocol Reference: Chow, S., 

Hedley, D., Grom, P., Magari, R., 

Jacobberger, J.W., and Shankey, 

T.V. (2005) Whole blood fixation 

and permeabilization protocol 

with red blood cell lysis for flow 

cytometry of intracellular phosphorylated 

epitopes in leukocyte 

subpopulations. Cytometry A. 

67, 4–17. 

PI/RNase Staining 

Solution labels DNA 

content without RNA 

crossreactivity. 

10 4 

10 3 

10 2 

10 1 

R-Phycoerythrin 

(PE) Conjugates 

For the most up-to-date list of ready 

to use flow validated antibodies conjugated 

to PE, eliminating the time and 

costs associated with PE conjugation 

and subsequent validation, go to 

www.cellsignal.com/peconjugates 

D. Immunostaining 

1. Aliquot 0.5–1x10 6 cells into each assay tube (by volume). 

2. Add 2 ml incubation buffer to each tube and wash by centrifugation. Repeat. 

3. Resuspend cells in 100 µl of diluted primary antibody prepared in incubation buffer at the recommended dilution. 

See individual antibody datasheet or product webpage for the appropriate dilution. 

4. Incubate for 1 hr at room temperature. 

5. Wash by centrifugation in 2 ml incubation buffer. 

6. If using a fluorochrome-conjugated primary antibody, resuspend cells in 0.5 ml 1X PBS and analyze on flow cytometer; 

for unconjugated or biotinylated primary antibodies, proceed to immunostaining (Step 9). 

7. Resuspend cells in fluorochrome-conjugated secondary antibody or fluorochrome-conjugated avidin, diluted in incubation 

buffer at the recommended dilution. 

8. Incubate for 30 min at room temperature. 

9. Wash by centrifugation in 2 ml incubation buffer. 

10. Resuspend cells in 0.5 ml 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA 

dye (Section E). 

E. Optional DNA Dye 

1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087). 

2. Incubate for at least 30 min at room temperature. 

3. Analyze cells in DNA staining solution on flow cytometer. 

Flow Cytometry Alternate Protocol 

(for combined staining of intracellular proteins and cell surface markers in blood) 


NOTE: Prepare solutions with reverse osmosis deionized or equivalent grade water. 

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2 

O, mix. 

2. 16% Formaldehyde (methanol free) 

3. Triton X-100: To prepare 50 ml of 0.1% Triton X-100 add 50 µl Triton X-100 to 50 ml 1X PBS and mix well. 

4. 50% methanol 

5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C. 

6. Secondary Antibodies: Anti-mouse (#4408, #8890, #4410, #8887), Anti-rabbit (#4412, #8889, #4414, #8885), Anti-rat 

(#4416, #4418) 

Fluorochrome 

Reference Table 

Max. excitation 

wavelength (nm) 

Max. emission 

wavelength (nm) 

Excitation 

laser line (nm) 

Color 

Brilliant Violet 421 407 421 405 violet 

DAPI 345 455 360/405 blue 

Pacific Blue 404 456 360/407 blue 

Hoechst 355 465 360 blue 

Alexa Fluor ® 488 495 519 488 green 

FITC 495 519 488 green 

Pacific Orange 403 551 360/407 orange 

Cy 3 548 561 488/532/561 orange 

Alexa Fluor ® 555 555 565 488/532/561 orange 

Phycoerythrin (PE) 496/566 576 488/532/561 orange 

Texas Red ® 595 613 568/595 red 

PE-Texas Red ® 496/566 616 488/532 red 

Alexa Fluor ® 594 590 617 561/595 red 

Propidium iodide (PI) 536 617 488/532 red 

7-AAD 546 647 488/532 red 

Allophycocyanin (APC) 650 660 633/635 red 

Alexa Fluor ® 647 650 665 633/635 red 

Cy 5 647 665 633/635 red 

PE-Cy 5 496/546 666 488/532/561 red 

PerCP 482 678 488/532 red 

DRAQ7 599/644 678/694 635/647 red 

DRAQ5 ® 647 681/697 488/633/647 red 

PE-Cy 5.5 565 693 488 red 

APC-Cy 5.5 650 694 633/635/647 red 

PerCP-Cy ® 5.5 482 695 488 red 

DyLight 680 692 712 680/685 red 

Alexa Fluor ® 700 696 719 633/635 near-IR 

APC-Cy 7 650 785 633/635 near-IR 

PE-Cy 7 496/566 785 488/532/561 near-IR 

DyLight 800 777 794 785 near-IR 

Alexa Fluor ® 790 784 814 785 near-IR 

10 0 0 200 400 600 800 1000 

DNA (PI) 

Propidium Iodide (PI)/RNase Staining 

Solution #4087: Flow cytometric analysis 

of Jurkat cells using Cyclin B1 (D5C10) 

XP ® #12231 Rabbit mAb and #4087 (DNA 

content). Anti-rabbit IgG (H+L), F(ab’) 2 

Fragment (Alexa Fluor ® 488 Conjugate) 

#4412 was used as a secondary antibody. 



205

11 


Immunofluorescence (IF) 

Fluorescent analysis of cells and tissues 

Immunofluorescence (IF) is a detection method that employs primary antibodies to reveal the localization 

of target proteins and analyze their relative expression levels in cultured cells or tissue sections. Primary 

antibodies can be directly labeled with a fluorescent tag (direct IF) or detected using a fluorescently 

labeled secondary antibody (indirect IF). The ability to detect multiple fluorescent tags as distinct colors 

within the same sample makes IF a powerful tool for investigating changes in subcellular localization, 

because protein location can be analyzed in relation to other labeled organelle markers or other cellular 

structures. 

Evaluate fixation and permeabilization 

protocols to achieve optimal staining. 

Cell and tissue samples may be preserved using either crosslinking or denaturing fixatives. (Note: not all 

samples are fixed for IF staining, such as in live cell staining.) The optimal fixation method is dependent 

upon the target protein, its subcellular localization, and in some cases the antibody that is used. Intracellular 

targets also require an additional permeabilization step. Detergents, such as Triton X-100 or Tween ® 

20 work well for most antibodies but in some cases, methanol permeabilization may be necessary. 

Optimizing fixation reagents can substantially improve staining results. 

Formaldehyde 

Methanol 

chapter 11: Immunofluorescence (IF) 

Methanol 

permeabilization 

improves intracellular 

immunostaining for 

some proteins. 

Triton X-100 

A number of factors can impact the ability 

to achieve bright and specific IF staining. 

• Antibody specificity and sensitivity • Target expression level • Species considerations 

• Permeabilization agent used • Type and degree of fixation • Dye selection 

• Antibody concentration and diluent • Cellular location of target 

• Detection method (direct, indirect, amplified) 

Keratin 8/18 (C51) Mouse mAb 

#4546: IF analysis of HeLa cells, 

fixed with formaldehyde (left) or 

methanol (right), using #4546 

(green). Red = Propidium Iodide 


Best with methanol fixation 

Methanol 

Nanog (D2A3) XP ® Rabbit mAb 

(Mouse Specific) #8822: Confocal 

IF analysis of F9 cells (Nanog positive) 

(A) and NIH/3T3 cells (Nanog negative) 

(B) was performed using #8822 

and competitor antibodies at the 

indicated concentrations. 

IF Tips for Success 

Use a well-characterized and specific 

antibody for accurate IF results. 

The primary antibody is an important reagent in successful IF experiments, because the specificity and 

sensitivity of the antibody dictates the quality of experimental data. 

Specificity of Nanog antibody is demonstrated by positive staining restricted 

to the nucleus and by absence of staining in a Nanog-negative cell line. 

A 

B 

CST #8822 

(0.25 µg/ml) 

Demonstrates specific nuclear 

staining in Nanog-expressing cells 

Crosslinking Fixatives 

Examples: formaldehyde, formalin, glutaraldehyde, glyoxal 

Note: Degree of crosslinking is dependent upon fixative 

concentration, temperature, and time of fixation. 

Advantages 

• Excellent tissue morphology 

AIF (D39D2) XP ® Rabbit mAb 

#5318: IF analysis of HeLa cells, 

fixed with formaldehyde (left) or 

methanol (right), using #5318 

(green). Red = Propidium Iodide 


Best with formaldehyde fixation 

• Inactivate enzymes such as proteases and phosphatases 

Limitations 

• Toxicity risk to user 

• Old formaldehyde solutions may induce autofluorescence 

PDI Antibody #2446: Confocal IF analysis 

of NIH/3T3 cells, permeabilized with 

0.3% Triton X-100 (top) or methanol 

(bottom), using #2446 (green) and 

β-Actin (8H10D10) Mouse mAb #3700 



Competitor antibodies used in accordance with manufacturer recommendations. 

Competitor 1 

(5.0 µg/ml) 

Shows cytoplasmic and nuclear staining 

in both positive and negative cell lines 

Competitor 2 

(2.0 µg/ml) 

Demonstrates some staining in the 

negative control and requires an 

8-fold higher IgG concentration 

Denaturing/Precipitating Fixatives 

Examples: ethanol, methanol, acetone, acetic acid 

Note: Denaturing fixatives may be combined with acetic 

acid to enhance tissue penetration. 

Advantages 

• Loss of soluble proteins can allow presentation of antigens normally inaccessible to antibodies 

• Work well with DNA stains 

• Do not require further permeabilization 

Limitations 

• May induce cell swelling or lysis, if not used cold 

• Loss of tertiary structure 

• Not advisable for use with CD markers 

• Protein loss may occur because of lack of crosslinking 

Permeabilization Agents 

For use after crosslinking fixatives, allows antibodies to penetrate cells and bind to intracellular antigens 

Detergent Examples: Triton X-100, Tween ® , Saponin, 

DOTMAC, SDS 

Alcohol Examples: ethanol, methanol 

Note: Alcohol permeabilization after fixation is not the 

same as alcohol fixation. 

• Typically not used with activation state-specific antibodies such as phospho-specific antibodies 

Detergent 

• Detergent permeabilization may require a separate step or may be included in blocking solution 

and antibody diluent (depending on the detergent used). 

Alcohol 

• Alcohol permeabilization may be required for some proteins. 


www.cellsignal.com/cstif 

207



Perform a titration to determine 

optimal antibody concentration. 

It is important to use an antibody at its optimal concentration. Using a lower concentration (more 

dilute) will diminish positive signal, but just as importantly, using higher concentration (less dilute) will 

increase background and decrease the signal-to-noise ratio. CST scientists routinely perform titrations 

using positive and negative cell lines to identify the concentration that gives optimal signal with minimal 

background staining. This recommended antibody dilution information is determined and provided with 

every lot of an IF-validated antibody. 

Antibody titration curves determine optimal concentration 

for maximal signal-to-noise in IF results. 

A 

Mean Fluorescence Intensity 

1200 

1000 

800 

600 

400 

200 

Optimal concentration for highest 

signal-to-noise with minimal background. 

50.0 

45.0 

40.0 

35.0 

30.0 

25.0 

20.0 

15.0 

10.0 

5.0 

Signal-to-Noise 

Choose a detection method that suits your 

target protein abundance and sample type. 

Conjugated primary antibodies (direct detection) offer convenience and a shorter protocol. Secondary 

antibodies conjugated to fluorochromes (indirect detection) may provide stronger signal intensity 

because multiple secondary antibodies can bind each primary antibody, but require using primary 

antibodies from different species when examining multiple targets within the same sample. Depending 

on the target expression level and sample type, direct and indirect IF may still be too dim for some 

assays, necessitating further amplification, such as avidin/biotin or tyramide. Avidin/biotin can improve 

signal intensity, but it may not be possible to completely block all nonspecific signal from endogenous 

biotin. More dramatic signal amplification can be achieved using tyramide amplification, which utilizes 

HRP-conjugated secondary antibodies to catalyze the deposition of fluorochrome-conjugated tyramide 

around the target. Tyramide amplification is very useful when performing IF in formalin-fixed paraffinembedded 

(FFPE) tissues. Signal amplification helps distinguish true signal from autofluorescence and 

negates issues related to antigen quality or scarcity, from either unmasking or protein loss. Tyramide 

also enables multiplex staining with antibodies from the same species (1). 

Retina stained with two directly conjugated 

antibodies from the same species 

Synapsin-1 (D12G5) XP ® Rabbit mAb (Alexa Fluor ® 594 Conjugate) #13556: 

Confocal IF analysis of rat retina using #13556 (red) and Neurofilament-L (C28E10) 

Rabbit mAb (Alexa Fluor ® 488 Conjugate) #8024 (green). Blue pseudocolor = 


0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 

Concentration (µg/ml) 

MFI (+) MFI (–) Signal-to-Noise 

B 

Dye-conjugated 

secondary antibodies 

can improve signal 

intensity for low 

abundance protein 

targets. 

Neurofilament-L (C28E10) Rabbit 


neuroepithelial clusters differentiated 

from human iPS cells, using #2837 detected 

with Anti-rabbit IgG (H+L), F(ab’) 2 

Fragment (Alexa Fluor ® 555 Conjugate) 

#4413 (blue) and β3-Tubulin (TU-20) 

Mouse mAb #4466 detected with 

Anti-mouse IgG (H+L), F(ab’) 2 Fragment 

(Alexa Fluor ® 488 Conjugate) #4408 



C 

Amplification enables detection of low abundance 

protein targets in paraffin-embedded tissue. 

0.031 µg/ml 0.063 µg/ml 0.125 µg/ml 0.25 µg/ml 1 µg/ml 

MUC1 (D9O8K) XP ® Rabbit mAb #14161: Graph depicting Mean Fluorescence Intensity (MFI) of ZR-75 cells (MUC1 expressing) and 

HCT 116 cells (MUC1 negative) using #14161, and calculated signal-to-noise (A). IF analysis of ZR-75 cells (B) and HCT 116 cells (C) at 

varying concentrations, as indicated, using #14161. Red = Propidium Iodide (PI)/RNase Staining Solution #4087. 

A 

B 

C 

E-Cadherin (24E10) Rabbit mAb 

#3195: IF analysis of FFPE human 

metastatic lymph node using #3195 

detected with a conjugated secondary 

antibody (A,B) or detection with Antirabbit 

IgG, HRP-linked Antibody #7074 

and a FITC-tyramide conjugate (C). 

Tissue Autofluorescence 

Low Exposure 

High Exposure 

Low Exposure 

Organelle Marker Samplers 

Organelle Marker Samplers provide a convenient collection of primary antibodies targeting well-established 

organelle associated proteins. For the most up-to-date listing of Organelle Marker Samplers, please go to 

www.cellsignal.com/organelles 

References: 

1. Toth, Z.E. and Mezey, E. (2007) J. Histochem. Cytochem. 55, 545–554. 


www.cellsignal.com/cstif 

209


Cultured Cells (Immunocytochemistry, IF-IC) Protocol 

IMPORTANT: Please refer to the Applications section on the front page of the product’s datasheet or product webpage to 

determine if this product is validated and approved for use on cultured cell lines (IF-IC), frozen tissue sections (IF-F), or paraffinembedded 

samples (IF-P). Some of CST’s antibodies perform optimally using an alternate protocol. Please consult the datasheet 

or webpage for product-specific recommendations. 


NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water. 


NOTE: Adjust pH to 8.0. 

2. Formaldehyde, 16%, methanol free: (Polysciences, Inc., cat #18814), use fresh, store opened vials at 4°C in dark. 

Dilute 1 in 4 in 1X PBS to make a 4% formaldehyde solution. 

3. Blocking Buffer: (1X PBS/5% normal serum/0.3% Triton X-100): To prepare 10 ml, add 0.5 ml normal serum from the 

same species as the secondary antibody (e.g., Normal Goat Serum (#5425) and 0.5 ml 20X PBS to 9 ml dH 2 O) and mix 

well. While stirring, add 30 µl Triton X-100. 

4. Antibody Dilution Buffer: (1X PBS/1% BSA/0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton X-100 to 10 ml 

1X PBS. Mix well then add 0.1g BSA (#9998), mix until well dissolved. 

5. Fluorochrome-conjugated Secondary Antibodies: (Anti-mouse #4408, #4409, #8890, #4410) (Anti-rabbit #4412, #4413, 

#8889, #4414) (Anti-rat #4416, #4417, #4418) 

6. Mounting Medium: ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961) 

B. Specimen Preparation 

I. Cultured Cell Lines (IF-IC) 

NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips. 

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS. 

NOTE: Formaldehyde is toxic, use only in a fume hood. 

2. Allow cells to fix for 15 min at room temperature. 

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each. 

4. Proceed with Immunostaining (Section C). 

C. Immunostaining 

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or 

covered dish/plate to prevent drying and fluorochrome fading. 

1. Block specimen in blocking buffer for 60 min. 

2. While blocking, prepare primary antibody by diluting as indicated on the product’s datasheet or webpage in antibody dilution 

buffer. 

3. Aspirate blocking solution, apply diluted primary antibody. 

4. Incubate overnight at 4°C. 

5. Rinse 3 times in 1X PBS for 5 min each. NOTE: If using a fluorochrome-conjugated primary antibody, proceed directly to 

Step 8. 

6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room 

temperature in the dark. 

7. Rinse 3 times in 1X PBS for 5 min each. 

8. Coverslip slides with ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961). 

9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C 

protected from light. 

Frozen/Cryostat Tissue Sections (IF-F) Protocol 

IMPORTANT: Please refer to the Applications section on the front page of the product’s datasheet to determine whether an 

antibody is validated and approved for use on cultured cell lines (IF-IC), frozen tissue sections (IF-F), or paraffin-embedded 

samples (IF-P). Some of CST’s antibodies perform optimally using an alternate protocol. Please consult the datasheet or webpage 

for product-specific recommendations. 



1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS, add 50 ml 20X PBS to 950 ml dH 2 O, mix. 

NOTE: Adjust pH to 8.0. 

2. Formaldehyde: 16%, methanol free: (Polysciences, Inc., cat #18814) Use fresh and store opened vials at 4°C in dark. 

Dilute in 1X PBS to make a 4% formaldehyde solution. 

3. Blocking Buffer (1X PBS/5% normal serum/0.3% Triton X-100): To prepare 10 ml, add 0.5 ml normal serum from 

the same species as the secondary antibody (e.g., Normal Goat Serum (#5425) and 0.5 ml 20X PBS to 9 ml dH 2 O and mix 

well. While stirring, add 30 µL Triton X-100. 

4. Antibody dilution buffer (1X PBS/1% BSA/0.3% Triton X-100): To prepare 10 ml, add 30 µL Triton X-100 to 10 ml 

1X PBS. Add 0.1 g BSA (#9998) and mix well until dissolved. 

5. Fluorochrome-conjugated Secondary Antibodies: Anti-mouse (#4408, #4409, #8890, #4410), Anti-rabbit (#4412, 

#4413, #8889, #4414), Anti-rat (#4416, #4417, #4418) 


B. Specimen Preparation 

1. For fixed frozen tissue, allow sections to air dry on the bench for 10 min, then proceed with Immunostaining (Section C). 

2. For fresh, unfixed frozen tissue, fix immediately as follows: 

a. Cover sections to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS. NOTE: Formaldehyde is toxic, use only 

in a fume hood. 

b. Allow sections to fix for 15 min at room temperature. 

c. Aspirate liquid, rinse slides 3 times in 1X PBS for 5 min each. 

d. Proceed with Immunostaining (Section C). 

C. Immunostaining 

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box 

or covered dish/plate to prevent drying and fluorochrome fading. 

1. Block specimen in Blocking Buffer for 60 min. 

2. While blocking, prepare primary antibody by diluting as indicated on the product’s datasheet or webpage in antibody dilution 

buffer. 

3. Aspirate Blocking Buffer, then apply diluted primary antibody. 


5. Rinse 3 times in 1X PBS for 5 min each. NOTE: If using a fluorochrome-conjugated primary antibody, proceed directly to 

Step 8. 

6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1 hr in the dark. 

7. Rinse 3 times in 1X PBS for 5 min each. 

8. Mount slides with ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961). 




Phalloidin labels actin filaments 

and DRAQ5 ® labels DNA. 

Alexa Fluor ® 555 Phalloidin #8953: Confocal IF analysis of HeLa cells using COX IV 

(3E11) Rabbit mAb (Alexa Fluor ® 488 Conjugate) #4853 (green). Actin filaments were 

labeled with #8953 (red). Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). 

ProLong ® Gold Antifade Reagent 

extends fluorescent signal, allowing 

more time to capture images. 

ProLong ® Gold Antifade Reagent #9071: Confocal IF analysis of SNB19 cells using 

β–Actin (8H10D10) Mouse mAb #3700 (red), Vimentin (D21H3) XP ® Rabbit mAb (Alexa 

Fluor ® 488 Conjugate) #9854 (green), and COX IV (3E11) Rabbit mAb (Alexa Fluor ® 647 

Conjugate) #7561 (blue). 



211


Paraffin Tissue Sections (IF-P) Protocol 

IMPORTANT: Please refer to the Applications section on the front page of the product’s datasheet to determine whether an antibody 

is validated and approved for use on cultured cell lines (IF-IC), frozen tissue sections (IF-F), or paraffin-embedded samples 

(IF-P). Some of CST’s antibodies perform optimally using an alternate protocol. Please consult the datasheet or webpage for 

product-specific recommendations. 


NOTE: Prepare solutions with reverse osmosis deionized or equivalent grade water (dH 2 O). 

1. Xylene 

2. Ethanol, anhydrous denatured, histological grade (100% and 95%) 

3. Antigen Unmasking: 

a. 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g sodium citrate trisodium salt dihydrate (C 6 H 5 Na 3 O 7 •2H 2 O) 

to 1 L dH 2 O. Adjust pH to 6.0. 

b. 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C 10 H 14 N 2 O 8 Na 2 • 2 H 2 O) to 1 L dH 2 O. Adjust pH to 8.0. 

4. 10X Tris Buffered Saline with Tween ® 20 (TBST): (#9997) To prepare 1 L 1X TBST, add 100 ml 10X TBST to 900 ml 

dH 2 O, mix. 

5. Incubation Buffer (1X TBST/5% normal serum): To prepare 10 ml, add 0.5 ml normal serum from the same species as 

the secondary antibody (e.g., Normal Goat Serum (#5425) to 9.5 ml of 1X TBST, mix well. 

6. Fluorochrome-conjugated Secondary Antibodies: Anti-mouse (#4408, #4409, #8890, #4410), Anti-rabbit (#4412, 

#4413, #8889, #4414), Anti-rat (#4416, #4417, #4418) 


B. Deparaffinization/Rehydration 

NOTE: Do not allow slides to dry at any time during this procedure. 

1. Incubate sections in 3 washes of xylene for 5 min each. 

2. Incubate sections in 2 washes of 100% ethanol for 10 min each. 

3. Incubate sections in 2 washes of 95% ethanol for 10 min each. 

4. Wash sections 2 times in dH 2 O for 5 min each. 

C. Antigen Unmasking 

NOTE: Consult product datasheet for specific recommendation for the unmasking solution/protocol. 

1. For Citrate: Bring slides to a boil in 10 mM Sodium Citrate Buffer, pH 6.0; maintain at a sub-boiling temperature (95–98°C) 

for 10 min. Cool slides on bench top for 30 min. 

2. For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0. Follow with 15 min at a sub-boiling temperature (95–98°C). No 

cooling is necessary. 

D. Immunostaining 

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box 

or covered dish/plate to prevent drying and fluorochrome fading. 

1. Wash sections 3 times in 1X TBST for 5 min each. 

2. Block specimen in Incubation Buffer for 60 min. 

3. While blocking, prepare primary antibody by diluting as indicated on the product’s datasheet or webpage in Incubation Buffer. 

4. Aspirate buffer from sections; apply diluted primary antibody. 


6. Wash 3 times in 1X TBST for 5 min each. NOTE: If using a fluorochrome-conjugated primary antibody, proceed directly to 

Step 9. 

7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Incubation Buffer for 1 hr at room temperature 

in the dark. 

8. Rinse 3 times in 1X TBST for 5 min each. 

9. Mount slides with ProLong ® Gold Antifade Reagent (#9071) or ProLong ® Gold Antifade Reagent with DAPI (#8961). 



In-Cell Western Protocol 


NOTE: Prepare solutions with reverse osmosis or equivalently purified water (dH 2 O). 

1. 10X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS add 80 g sodium chloride (NaCl), 2 g potassium chloride 

(KCl), 14.4 g sodium phosphate, dibasic (Na 2 HPO 4 ) and 2.4 g potassium phosphate, monobasic (KH 2 PO 4 ) to 1 L dH 2 O. Adjust 

pH to 7.4. 

2. Formaldehyde, 16%, methanol-free: (Polysciences, Inc. #18814) Use fresh; store opened vials at 4°C in dark, dilute in 

1X PBS for use. 

3. Blocking Buffer: To prepare 25 ml, add 2.5 ml 10X PBS, 1.25 ml normal serum from the same species as the secondary 

antibody (e.g., normal goat serum (#5425), normal donkey serum) and 21.25 ml dH 2 O and mix well. While stirring, add 75 µl 

Triton X-100 (100%). 

4. Antibody Dilution Buffer: To prepare 40 ml, add 4 ml 10X PBS to 36 ml dH 2 O, mix. Add 0.4 g BSA and mix well. While 

stirring, add 120 µl Triton X-100 (100%). 

5. Fluorochrome-conjugated secondary antibody. 

NOTE: When using any primary or fluorochrome-conjugated secondary antibody for the first time, titrate the antibody to 

determine which dilution allows for the strongest specific signal with the least background for your sample. 

B. Fixation and Immunolabeling 

NOTE: Please refer to the IF protocol for each antibody to identify the recommended antibody-specific fixation and permeabilization 

conditions. Cells should be grown, treated, fixed, and stained directly in multi-well plates. To avoid edge effects from uneven 

distribution of cells in individual wells, let newly-seeded plates stand at room temperature for one hour before placing in 37°C 

incubator (for more information, see Lundholt, B.K., Scudder, K.M., and Pagliaro, L. (2003) A simple technique for reducing 

edge effect in cell-based assays. J. Biomol. Screen. 8, 566–570). To minimize false signal from scattered light and background 

fluorescence, use black-walled multi-well plates (e.g., BD Falcon, #353948). Avoid touching the inside of the wells with pipettes 

or aspirators as this may leave an artifact that will affect subsequent quantification. Instead, empty wells by shaking inverted 

plate sharply over waste receptacle and then blotting with clean paper towel. Volumes indicated below are for use with standard 

96-well plates. Adjust volume accordingly for plates with larger or smaller wells. 

1. Grow and treat cells as desired in multi-well plates. 

2. Fix cells with 50 µl 4% formaldehyde or 100% methanol, as recommended in the antibody-specific IF protocol. 

3. Allow cells to fix for 15 min at room temperature. 

4. Shake inverted plate over aldehyde waste receptacle and blot with clean paper towel. 

5. Rinse plate 3 times for 5 min each with room temperature PBS (100 µl/well). 

6. Block cells in Blocking Buffer for one hour at room temperature (50 µl/well). 

7. While blocking, prepare primary antibody by diluting in Antibody Dilution Buffer as indicated for Immunofluorescence (IF-IC) 

on datasheet. 

8. Shake out blocking solution and add diluted primary antibody (total volume 50 µl/well). 

NOTE: For double-labeling, prepare a cocktail of mouse and rabbit primary antibodies at their appropriate dilutions in 

Antibody Dilution Buffer. 

9. Cover plate and incubate overnight at 4°C. 

10. Rinse plate 3 times in PBS for 5 min each. 

11. Add fluorochrome-conjugated secondary antibody* diluted in Antibody Dilution Buffer (total volume 50 µl/well) and incubate 

for 1 hr at room temperature in the dark. 

NOTE: For double-labeling, prepare a cocktail of fluorochrome-conjugated anti-mouse and anti-rabbit secondary antibodies 

in Antibody Dilution Buffer. 

12. Rinse plate 3 times in PBS for 5 min each. 

13. To normalize for cell number, label nuclei using DRAQ5 ® diluted 1:1000 in PBS (total volume 50 µl/well). Incubate at least 

30 min at room temperature in the dark prior to scanning. 

NOTE: DRAQ5 ® can be emptied and the wells rinsed at least 1X with PBS for better optical resolution. 

14. Scan plate according to manufacturer’s directions. (Be sure bottom of plate has been wiped clean prior to scanning). 

Recommended Secondary Antibodies: Anti-Rabbit: Anti-Rabbit IgG (H+L) (DyLight ® 680 Conjugate) #5366, Anti-Rabbit IgG 

(H+L) (DyLight ® 800 Conjugate) #5151, Anti-Mouse: Anti-Mouse IgG (H+L) (DyLight ® 680 Conjugate) #5470, Anti-Mouse IgG 

(H+L) (DyLight ® 800 Conjugate) #5257 


IF Companion Products 

See our comprehensive list of IF companion products that are validated in-house with our protocols so that you can 

get the reliable reagents you need to complete your experiment. www.cellsignal.com/ifcompanion 



213

12 


chapter 

Antibodies targeting 

the same protein 

may have different 

preferred methods 

of antigen retrieval. 

EGF Receptor (D38B1) XP ® Rabbit 

mAb #4267: IHC analysis of paraffinembedded 

human lung carcinoma 

using #4267 and an EGFR mouse 

mAb after antigen retrieval by boiling 

in citrate buffer (left), boiling in EDTA 

buffer (center), or digestion with 

pepsin (right). 

For #4267, superior signal is 

obtained with EDTA retrieval. 

However, for the competitor’s EGFR 

mouse mAb, signal is only achieved 

with pepsin digestion. 

Immunohistochemistry (IHC) 

Tissue Analysis 

Immunohistochemistry (IHC) is a common technique for morphological characterization of tumors or other 

tissue malignancies. IHC uses antibodies to detect and analyze protein expression while maintaining 

the composition, cellular characteristics, and structure of native tissue. Starting with high quality tissue 

samples is important for successful IHC results, so it is best to use freshly cut sections. It is also essential 

to include appropriate controls to assess antibody performance. Prior to testing on tissue, antibody 

specificity can be evaluated at the cellular level using a variety of cell lines and treatment conditions. 

• Total protein specificity can be assessed through the use of positively and negatively expressing 

cell lines. 

• Cells can be treated with biological or chemical modulators known to induce signaling changes 

to verify modification specificity, such as phosphorylation, acetylation, cleavage, etc. 

• Phospho-specific antibodies can be evaluated with phosphatase treatment. 

• Isotype control antibodies help rule out nonspecific staining of primary antibodies due to Fc 

receptor binding or other protein-protein interactions and should have the same immunoglobulin 

type as the test antibody. 

The IHC protocol includes variations in antigen retrieval methods, antibody diluents, and detection 

systems; the optimal method for these parameters must be determined experimentally for each 

primary antibody in order to produce the maximum signal. 

The most common problems encountered when performing IHC are weak specific staining or high 

background. Some challenges, such as incomplete or poor antigen retrieval, are broadly applicable to 

most antibodies, while other challenges are specific to certain species or protein modifications. 

IHC Tips for Success 

Antigen retrieval optimization can greatly improve staining. 

The crosslinks created during the fixation step can prevent antibody binding by inhibiting access to the 

antigen; therefore, it is important to reverse crosslinks using a procedure called antigen retrieval (also 

known as antigen unmasking or epitope retrieval). Antigen retrieval can be achieved through either 

a heat-induced method (heat-induced epitope retrieval; HIER) or through enzymatic digestion, and 

the optimal form of retrieval is specific for the unique antibody/antigen and not for the protein itself. 

Therefore, if you use more than one antibody against a particular protein target, the optimal retrieval 

conditions for each antibody must be determined individually. 


Competitor’s mAb 

Citrate EDTA Pepsin 

Avoid casein-based solutions when 

using a phospho-specific antibody. 

Blocking is an important step in the IHC protocol because it prevents nonspecific background staining. 

Commercially available blocking solutions that contain casein should be avoided when working with 

phospho-specific primary antibodies as they tend to diminish signal intensity. 

Phospho-Histone H2A.X (Ser139) 

(20E3) Rabbit mAb #9718: IHC 


breast carcinoma using #9718 after 

blocking with normal goat serum 

(NGS) (left) or a casein-based blocking 

solution (right). 

TBST/5% NGS 

Avoid same-species background. 

Casein 

When the primary antibody is from the same species as the sample being tested, the secondary antibody 

may bind endogenous IgG in some tissues, causing high background (mouse-on-mouse staining, 

for example). Including a control slide stained without the primary antibody can establish whether or 

not the secondary antibody is the source of the background. 

Ki-67 (D3B5) Rabbit mAb (Mouse 

Specific; IHC Formulated) #12202: 

IHC analysis of paraffin-embedded 

mouse lung using #12202 (left) or 

a competitor’s Ki-67 mouse mAb 

(right). Analysis with #12202 displays 

only nuclear staining, while analysis 

with the mouse mAb shows high 

background staining. 


Competitor’s mouse mAb 

Use a polymer-based detection system to increase sensitivity 

and avoid endogenous biotin background staining. 

Traditional IHC detection methods use a biotinylated secondary antibody followed by exposure to an 

avidin-HRP complex prior to chromogenic detection. Biotin-based systems are prone to background 

staining, particularly in tissues that possess high levels of endogenous biotin, such as liver and kidney. 

Polymer-based detection reagents consist of enzymes and secondary antibodies directly conjugated to 

a polymer backbone. These systems eliminate false-positive staining due to endogenous biotin. The 

polymer-based system also improves sensitivity, enabling detection of low abundance targets. Signal- 

Stain ® Boost IHC Detection Reagents save time by eliminating one step in the detection procedure and 

are compatible with all peroxidase-based substrates. 

SignalStain ® Boost IHC Detection 

Reagent #8114: IHC analysis of 

paraffin-embedded human lung carcinoma 

using Sox2 (D6D9) XP ® Rabbit 

mAb #3579 and either biotin-based 

detection (left) or polymer-based 

detection #8114 (right). 


Biotin-based Detection 

Polymer-based Detection 

12: Immunohistochemistry (IHC) 

Casein block 

produces a lower 

overall signal when 

compared with 

TBST/5% NGS. 

When possible, 

avoid using primary 

antibodies raised in 

the same species as 

the tissue examined. 

Polymer-based 

detection is more 

sensitive than biotinbased 

systems. 



www.cellsignal.com/cstihc 

215


chapter 12: Immunohistochemistry (IHC) 

IHC Tips & 

Techniques 

Videos 

For more in-depth help with IHC, 

please see our IHC Protocols and 

Troubleshooting videos at 

www.cellsignal.com/ihcvideo 

IHC Paraffin Protocol (using SignalStain ® Boost Detection Reagent) 

*IMPORTANT: Please refer to the Applications section on the front page of product datasheet or product webpage to determine 

whether a product is validated and approved for use on paraffin-embedded (IHC-P) tissue sections. Please see product datasheet 

or product webpage for appropriate antibody dilution and unmasking solution. 



1. Xylene 

2. Ethanol, anhydrous denatured, histological grade (100% and 95%) 

3. Deionized water (dH 2 O) 

4. Hematoxylin (optional) 

5. Wash Buffer: 1X Tris Buffered Saline with Tween ® 20 (TBST): To prepare 1 L 1X TBST: add 100 ml 10X TBST (#9997) to 

900 ml dH 2 O, mix. 

6. Antibody Diluent Options:* 

a. SignalStain ® Antibody Diluent: (#8112) 

b. TBST/5% normal goat serum: To 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425). 

c. PBST/5% normal goat serum: To 5 ml 1X PBST, add 250 µl Normal Goat Serum (#5425). 

1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 50 ml 20X PBS (#9808) to 950 ml dH 2 O, mix. 

1X PBS/0.1% Tween ® 20 (PBST): To prepare 1 L 1X PBST: add 1 ml Tween ® 20 to 1 L 1X PBS and mix. 

7. Antigen Unmasking Options:* 

a. Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate 

(C 6 H 5 Na 3 O 7 •2H 2 O) to 1 L dH 2 O. Adjust pH to 6.0. 

b. EDTA: 1 mM EDTA: To prepare 1 L add 0.372 g EDTA (C 10 H 14 N 2 O 8 Na 2 •2H 2 O) to 1 L dH 2 O. Adjust pH to 8.0. 

c. TE: 10 mM Tris/1 mM EDTA, pH 9.0: To prepare 1 L, add 1.21 g Tris base (C 4 H 11 NO 3 ) and 0.372 g EDTA 

(C 10 H 14 N 2 O 8 Na 2 •2H 2 O) to 950 ml dH 2 O. Adjust pH to 9.0, then adjust final volume to 1 L with dH 2 O. 

d. Pepsin: 1 mg/ml in Tris-HCl, pH 2.0. 

8. 3% Hydrogen Peroxide: To prepare 100 ml, add 10 ml 30% H 2 O 2 to 90 ml dH 2 O. 

9. Blocking Solution: TBST/5% Normal Goat Serum: to 5 ml 1X TBST, add 250 µl Normal Goat Serum (#5425). 

10. Detection System: SignalStain ® Boost IHC Detection Reagents (HRP, Mouse #8125; HRP, Rabbit #8114 ) 

11. Substrate: SignalStain ® DAB Substrate Kit (#8059). 

B. Deparaffinization/Rehydration 

NOTE: Do not allow slides to dry at any time during this procedure. 

1. Deparaffinize/hydrate sections: 

a. Incubate sections in 3 washes of xylene for 5 min each. 

b. Incubate sections in 2 washes of 100% ethanol for 10 min each. 

c. Incubate sections in 2 washes of 95% ethanol for 10 min each. 

2. Wash sections 2 times in dH 2 O for 5 min each. 

C. Antigen Unmasking* 

NOTE: Consult product datasheet for specific recommendation for the unmasking solution/protocol. 

1. For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. 

Cool slides on bench top for 30 min. 

2. For EDTA: Bring slides to a boil in 1 mM EDTA, pH 8.0: follow with 15 min at a sub-boiling temperature. No cooling 

is necessary. 

3. For TE: Bring slides to a boil in 10 mM Tris/1 mM EDTA, pH 9.0: then maintain at a sub-boiling temperature for 18 min. 

Cool at room temperature for 30 min. 

4. For Pepsin: Digest for 10 min at 37°C. 

D. Staining 

NOTE: Consult product datasheet for recommended antibody diluent. 

1. Wash sections in dH 2 O 3 times for 5 min each. 

2. Incubate sections in 3% hydrogen peroxide for 10 min. 


4. Wash sections in wash buffer for 5 min. 

5. Block each section with 100–400 µl blocking solution for 1 hr at room temperature. 

6. Remove blocking solution and add 100–400 µl primary antibody diluted in recommended antibody diluent to each section*. 

Incubate overnight at 4°C. 

7. Equilibrate SignalStain ® Boost Detection Reagent to room temperature. 

8. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 

9. Cover section with 1–3 drops SignalStain ® Boost Detection Reagent as needed. Incubate in a humidified chamber for 

30 min at room temperature. 


10. Wash sections three times with wash buffer for 5 min each. 

11. Add 1 drop (30 µl) SignalStain ® DAB Chromogen Concentrate to 1 ml SignalStain ® DAB Diluent and mix well before use. 

12. Apply 100–400 µl SignalStain ® DAB to each section and monitor closely. 1–10 min generally provides an acceptable 

staining intensity. 

13. Immerse slides in dH 2 O. 

14. If desired, counterstain sections with hematoxylin per manufacturer’s instructions. 

15. Wash sections in dH 2 O two times for 5 min each. 

16. Dehydrate sections: 

a. Incubate sections in 95% ethanol two times for 10 sec each. 

b. Repeat in 100% ethanol, incubating sections two times for 10 sec each. 

c. Repeat in xylene, incubating sections two times for 10 sec each. 

17. Mount sections with coverslips. 

For optimal results, always use the recommended primary 

antibody diluent, as indicated on the product datasheet. 

SignalStain ® Antibody Diluent 

#8112: IHC analysis of paraffinembedded 

human breast carcinoma 

(top) and HCC827 xenograft (bottom) 

using Phospho-Akt (Ser473) (D9E) 

XP ® Rabbit mAb #4060 or Phospho- 

EGF Receptor (Tyr1173) (53A5) Rabbit 

mAb #4407 after dilution in either 

#8112 (left) or TBST/5% NGS (right). 

A superior signal is achieved 

when #4060 is diluted in #8112 as 

compared with TBST/5% NGS. 

No one diluent enables the optimal 

performance of all antibodies. 

#4060 

#4407 

SignalStain ® #8112 

TBST-5% NGS 

IHC Frozen Protocol (using SignalStain ® Boost Detection Reagent) 

IMPORTANT: Please refer to the Applications section on the front page of product datasheet or product webpage to determine 

whether a product is validated and approved for use on frozen (IHC-F) tissue sections. Please see product datasheet or product 

webpage for appropriate antibody dilution. 

NOTE: Please see product datasheet and website for product-specific protocol recommendations. 



1. Xylene 

2. Ethanol (anhydrous denatured, histological grade 100% and 95%) 

3. Hematoxylin (optional) 

4. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 50 ml 20X PBS (#9808) to 950 ml dH 2 O, mix. 

5. Fixative Options: For optimal fixative, please refer to the product datasheet. 

a. 10% neutral buffered formalin 

b. Acetone 

c. Methanol 

d. 3% Formaldehyde: To prepare 100 ml, add 18.75 ml 16% formaldehyde to 81.25 ml 1X PBS. 

6. Wash Buffer: 1X Tris Buffered Saline with Tween ® 20 (TBS): To prepare 1 L 1X TBS: add 100 ml 10X TBS (#12498) to 900 

ml dH 2 O, mix. 

7. Methanol/Peroxidase: To prepare, add 10 ml 30% H 2 O 2 to 90 ml methanol. Store at -20°C. 

8. Blocking Solution: 1X TBS/0.3% Triton X-100/5% Normal Goat Serum (#5425). To prepare, add 500 µl goat serum and 

30 µl Triton X-100 to 9.5 ml 1X TBS. 

9. Detection System: SignalStain ® Boost IHC Detection Reagents (HRP, Mouse #8125; HRP, Rabbit #8114 ) 

10. Substrate: SignalStain ® DAB Substrate Kit (#8059). 


217


B. Sectioning 

1. For tissue stored at -80°C: Remove from freezer and equilibrate at -20°C for approximately 15 min before attempting to 

section. This may prevent cracking of the block when sectioning. 

2. Section tissue at a range of 6–8 µm and place on positively charged slides. 

3. Allow sections to air dry on bench for a few min before fixing (this helps sections adhere to slides). 

C. Fixation Options 

NOTE: Consult product datasheet to determine the optimal fixative. 

1. After sections have dried on the slide, fix in optimal fixative as directed below. 

a. 10% Neutral Buffered Formalin: 10 min at room temperature. Proceed with staining procedure immediately (Section D). 

b. Cold Acetone: 10 min at -20°C. Air dry. Proceed with staining immediately (Section D). 

c. Methanol: 10 min at -20°C. Proceed with staining immediately (Section D). 

d. 3% Formaldehyde: 15 min at room temperature. Proceed with staining immediately (Section D). 

e. 3% Formaldehyde/methanol: 15 min at room temperature in 3% formaldehyde, followed by 5 min in methanol at 

-20°C (do not rinse in between). Proceed with staining immediately (Section D). 

D. Staining 

1. Wash sections in wash buffer 2 times for 5 min. 

2. Incubate for 10 min at room temperature in methanol/peroxidase. 

3. Wash sections in wash buffer 2 times for 5 min. 

4. Block each section with 100–400 µl blocking solution for 1 hr at room temperature. 

5. Remove blocking solution and add 100–400 µl primary antibody diluted in blocking solution to each section. 


7. Equilibrate SignalStain ® Boost Detection Reagent to room temperature. 

8. Remove antibody solution and wash sections in wash buffer three times for 5 min each. 

9. Cover section with 1–3 drops SignalStain ® Boost Detection Reagent as needed. Incubate in a humidified chamber for 30 

min at room temperature. 

10. Wash sections 3 times with wash buffer for 5 min each. 

11. Add 1 drop (30 µl) SignalStain ® DAB Chromogen Concentrate to 1 ml SignalStain ® DAB Diluent and mix well before use. 

12. Apply 100–400 µl SignalStain ® DAB to each section and monitor closely. 1–10 min generally provides an acceptable 

staining intensity. 

13. Immerse slides in dH 2 O. 

14. If desired, counterstain sections with hematoxylin per manufacturer’s instructions. 


16. Dehydrate sections: 

a. Incubate sections in 95% ethanol 2 times for 10 sec each. 

b. Repeat in 100% ethanol, incubating sections 2 times for 10 sec each. 

c. Repeat in xylene, incubating sections 2 times for 10 sec each. 

17. Mount sections with coverslips. 

Not all DAB substrates perform equally. 


Guide to Successful IHC 

Competitor’s 


SignalStain ® DAB Substrate Kit #8059: 

IHC analysis of paraffin-embedded human 

breast carcinoma using Phospho-Stat3 

(Tyr705) (D3A7) XP ® Rabbit mAb #9145. 

Chromogenic detection was performed 

using #8059 (left) or DAB supplied by a 

competitor (right). 

Helpful tips and explanations to support your IHC experiments are available in “A Guide to Successful 

Immunohistochemistry”. Request a copy at www.cellsignal.com/ihcguide 

IHC Troubleshooting Guide 

Suboptimal IHC staining is frequently resolved by adjusting relatively few variables. Adjustments to key steps within the protocol, 

such as antigen retrieval, can often resolve these common issues. 

Little or no staining: 

Cause 

Sample storage 

Tissue sections dried out 

Slide preparation 

Antigen unmasking/retrieval 

Unmasking/retrieval buffer 

Antibody dilution/diluent 

Incubation time 

Detection system 

Negative staining 

High background: 

Cause 

Slide preparation 

Peroxidase quenching 

Biotin block 

Blocking 

Antibody dilution/diluent 

Secondary cross reactivity 

Washes 

Solution 

Slides may lose signal over time in storage. This process is variable and dependent upon 

the protein target. The effect of slide storage on staining has not been established for every 

protein; therefore, it is best practice that slides are freshly cut before use. If slides must be 

stored, do so at 4°C. Do not bake slides before storage. 

It is vital that the tissue sections remain covered in liquid throughout the staining procedure. 

Inadequate deparaffinization may cause spotty, uneven background staining. Repeat the 

experiment with new sections using fresh xylene. 

Fixed tissue sections have chemical crosslinks between proteins that, dependent on the 

tissue and antigen target, may prevent antibody access or mask antigen targets. Antigen 

unmasking protocols may utilize a hot water bath, microwave, or pressure cooker. Antigen 

unmasking protocols utilizing a water bath are not recommended. Antigen unmasking 

performed with a microwave is preferred, though staining of particular tissues or antigen 

targets may require the use of a pressure cooker. 

Staining of particular tissues or antigen targets may require an optimized unmasking buffer. 

Refer to product datasheet for antigen unmasking buffer recommendations. Always prepare 

fresh 1X solutions daily. 

Consult CST product datasheet for the recommended dilution and diluent. Titration of the 

antibody may be required if a reagent other than the one recommended is used. 

Primary antibody incubation according to a rigorously tested protocol provides consistent, 

reliable results. CST antibodies have been developed and validated for optimal results 

when incubated overnight at 4°C. 

Polymer-based detection reagents, such as SignalStain ® Boost IHC Detection Reagents 

(#8114 and #8125), in conjunction with SignalStain ® DAB Substrate Kit (#8059), are more 

sensitive than avidin/biotin-based detection systems. Standard secondary antibodies directly 

conjugated with HRP may not provide sufficient signal amplification. Always verify the 

expiration date of the detection reagent prior to use. 

A complete lack of staining may indicate an issue with the antibody or protocol. Employ a 

high expressing positive control, such as paraffin-embedded cell pellets, to ensure that the 

antibody and procedure are working as expected. 

Phospho-specific antibodies in particular, or any antibody directed against a rarely 

expressed protein, may not stain 100% of the cases of a given indication. It is possible that 

the sample is truly negative. 

Solution 

Inadequate deparaffinization may cause spotty, uneven background staining. Repeat the 

experiment with new sections using fresh xylene. 

Endogenous peroxidase activity in samples may produce excess background signal if an 

HRP-based detection system is being used. Quench slides in a 3% H 2 O 2 solution, diluted in 

RODI water, for 10 min prior to incubation with the primary antibody. 

Using biotin-based detection systems with samples that have high levels of endogenous 

biotin, such as kidney and liver tissues, may be problematic. In this case, use a polymerbased 

detection system such as SignalStain ® Boost IHC Detection Reagents (#8114 and 

#8125). A biotin block may also be performed after the normal blocking procedure prior to 

incubation in primary antibody. 

Block slides with 1X TBST (#9997) with 5% Normal Goat Serum (#5425) for 30 min prior to 

incubation with the primary antibody. 

Consult CST product datasheet for the recommended dilution and diluent. Titration of the 

antibody may be required if a reagent other than the one recommended is used. 

The secondary antibody may bind endogenous IgG, causing high background, in some 

samples where the secondary antibody is raised in the same species as the sample being 

tested (mouse-on-mouse staining). Include a control slide stained without the primary 

antibody to confirm whether the secondary antibody is the source of the background. 

Adequate washing is critical for contrasting low background and high signal. Wash slides 

three times for 5 min with TBST (#9997) after primary and secondary antibody incubations. 

chapter 12: Immunohistochemistry (IHC) 



219

13 


chapter 

Sandwich ELISA 

The enzyme-linked immunosorbent assay (ELISA) is a method for detecting a specific analyte in a 

heterogeneous biological fluid or cell extract. ELISA is a quantitative immunoassay typically perfomed 

in multi-well microplates, which allows for simultaneous analysis of multiple samples. In a sandwich 

ELISA, the target molecule is bound between a capture antibody immobilized on a microplate and a 

detection antibody that is detected by a secondary antibody conjugated to an enzymatic or other reporter 

system. The use of two antibodies recognizing different epitopes of the same protein results in a highly 

specific assay. Sandwich ELISA assays are useful for the screening of small molecule inhibitors without 

using expensive instrumentation or complex data analysis methods. Sandwich ELISA output can be 

measured by a number of optical methods, including colorimetric (absorbance) and chemiluminescent 

(RLU or relative light units). Chemiluminescent readouts show a higher dynamic range, especially at low 

target protein concentrations. The specifcity and sensitivity of the capture and detection antibodies are 

critical to ensuring high signal-to-background ratios. The capture and detection antibody pairs are also 

amenable to customization on plate-based and bead-based platforms. 

Rapid cell harvest and elevated phosphatase 

inhibitor concentrations may improve ELISA 

results for low abundance proteins. 

While it is important to achieve complete cell lysis for the investigation of both total and post-translationally 

modified protein targets, rigorous extraction methods must be balanced with preservation of 

the antibody-antigen interactions required for ELISA. Preserving the phosphorylation state of target 

epitopes can be more challenging when working with specific cell lines or tissues, as some cells 

produce higher levels of phosphatases than others. When investigating the phosphorylation state of 

low abundance proteins or targets that are difficult to detect, especially in high phosphatase-producing 

cell lines, it is sometimes necessary to replace the standard protocol for preparation of cell lysates 

with a rapid harvest method. This method omits the cell scraping and sonication steps and uses a lysis 

buffer that contains increased concentrations of Ser/Thr phosphatase inhibitors. For example, when 

initial ELISA results from phospho-targets produce a low signal, scientists at CST use a no scrape, no 

sonicate rapid harvest method combined with a lysis buffer (#7018) that contains increased amount of 

phosphatase inhibitors*, as a means to increase target-specific signal. 

13: Sandwich eLISA 

*Complete protocol and buffer components 

can be found on the PathScan ® 

Sandwich ELISA Lysis Buffer #7018 

product datasheet 

Improved ELISA detection 

of phospho-AMPKα (Thr172) 

was achieved using an 

alternative lysis buffer and 

a rapid extraction protocol. 

3 

2.5 

2 

1.5 

1 

0.5 

PathScan ® Phospho-AMPKα 

(Thr172) Sandwich ELISA Kit #7959: 

C2C12 cells, untreated or treated with 

H 2 0 2 (10 mM for 10 min), were lysed and 

harvested using Cell Lysis Buffer (10X) 

#9803 or PathScan ® Sandwich ELISA 

Lysis Buffer #7018 (which contains 

inhibitors of Ser/Thr phosphatases) and 

detected using #7959. Equal protein 

concentrations of cell extract were used 

in each assay. ELISA signal was greatest 

when cell lysis was performed with 

#7018 and a rapid harvest procedure 

with no cell scraping or sonication. 

0 

Scrape/Sonicate No Scrape/Sonicate Scrape/Sonicate No Scrape/Sonicate 

#9803 

#7018 

Cell Lysate total protein concentration (0.25 mg/ml) 

Untreated 

H 2 O 2 Treated 

Phosphatase 

inhibitors preserve 

the constitutive phosphorylation 

of Mer. 

ELISA Tips for Success 

For detection of intracellular targets, the success of an ELISA experiment depends critically on the 

quality and composition of the cell lysis buffer and the method used to generate the cell extract. 

Include protease and/or phosphatase inhibitors in lysis 

buffer to preserve protein levels and phosphorylation state. 

Using fresh cell lysis buffer containing appropriate protease and phosphatase inhibitors ensures that 

changes in the protein or phosphorylation levels are not due to proteolytic degradation of target proteins 

or loss of phosphorylation due to endogenous phosphatases. 


3 

2 

1 

0 

Total Mer 

Phosho-Mer (panTyr) 

– + – + 

phosphatase 

inhibitor 

PathScan ® Phospho-Mer (panTyr) Sandwich ELISA Kit #13340: Constitutive 

phosphorylation of Mer in HCC827 cells lysed in the presence of protease inhibitors 

and phosphatase inhibitors (phospho lysate) is detected by #13340 (upper, right). 

In contrast, a low level of phospho-Mer protein is detected in HCC827 cells lysed 

in the presence of protease inhibitors and absence of phosphatase inhibitors 

(nonphospho lysate). Similar levels of total Mer protein from both nonphospho and 

phospho lysates are detected by PathScan ® Total Mer Sandwich ELISA Kit #13488 

(upper, left). Absorbance at 450 nm is shown in the top figure while corresponding 

western blots using Mer (D21F11) XP ® Rabbit mAb #4319 (left) and a phospho-Mer 

(Tyr749/753) antibody (right) are shown in the bottom figure. Phosphatase inhibitors 

include sodium pyrophosphate, β-glycerophosphate, and sodium orthovanadate. 

Nonphospho 

Phospho 

PathScan ® Sandwich ELISA Colorimetric Protocol 

NOTE: Refer to product-specific datasheet or product webpage for assay incubation temperature. 


For lyophilized formulation 


1. Microwell strips: Bring all to room temperature before use. 

2. Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 1.0 ml of Detection Antibody Diluent (green 

solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully 

reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted Detection Antibody to 10.0 ml of 

Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C. 

3. HRP-Linked Antibody: Supplied lyophilized as a red colored cake or powder. Add 1.0 ml of HRP Diluent (red solution) to 

yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. 

To make the final working solution, add the full 1.0 ml volume of reconstituted HRP-Linked Antibody to 10.0 ml of HRP 

Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C. NOTE: Some PathScan ® 

ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody. 

4. Detection Antibody Diluent: Green colored diluent for reconstitution and dilution of the detection antibody (11 ml provided). 

5. HRP Diluent: Red colored diluent for reconstitution and dilution of the HRP‐Linked Antibody (11 ml provided). 

6. Sample Diluent: Blue colored diluent provided for dilution of cell lysates. 

7. 1X Wash Buffer: Prepare by diluting 20X Wash Buffer (included in each PathScan ® Sandwich ELISA Kit) in purified water. 

8. 1X Cell Lysis Buffer: Prepare 1X lysis buffer using either PathScan ® Sandwich ELISA buffer (ready to use 1X stock, #7018) 

or 10X Cell Lysis Buffer (#9803). To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml 10X Cell Lysis Buffer to 9 ml of dH 2 O, 

mix. Both buffers can be stored at 4°C for short-term use (1–2 weeks). Recommended: Add 1 mM phenylmethylsulfonyl 

fluoride (PMSF) (#8553) immediately before use. NOTE: Refer to product-specific datasheet or webpage for lysis buffer 

recommendation. 

9. TMB Substrate: (#7004) 

10. STOP Solution: (#7002) 


www.cellsignal.com/cstelisa 

221


chapter 13: Sandwich eLISA 

For liquid formulation 


1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L PBS: add 50 ml 10X PBS to 950 ml dH 2 O, mix. 

2. Bring all microwell strips to room temperature before use. 

3. Prepare 1X Wash Buffer by diluting 20X Wash Buffer (included in each PathScan ® Sandwich ELISA Kit) in dH 2 O. 

4. 1X Cell Lysis Buffer: 10X Cell Lysis Buffer (#9803): To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10X Cell Lysis 

Buffer to 9 ml of dH 2 O, mix. PathScan ® Sandwich ELISA Lysis Buffer (#7018) 1X: This buffer is ready to use as is. Both buffers 

can be stored at 4°C for short-term use (1–2 weeks). Recommended: Add 1 mM phenylmethylsulfonyl fluoride (PMSF) 

(#8553) immediately before use. NOTE: Refer to product-specific datasheet or webpage for lysis buffer recommendation. 



B. Preparing Cell Lysates 

For adherent cells 

1. Aspirate media when the culture reaches 80–90% confluence. Treat cells by adding fresh media containing regulator for 

desired time. 

2. Remove media and rinse cells once with ice-cold 1X PBS. 

3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the 

plate on ice for 5 min. 

4. Scrape cells off the plate and transfer to an appropriate tube. Keep on ice. 

5. Sonicate lysates on ice. 

6. Microcentrifuge for 10 min (x14,000 rpm) at 4°C and transfer the supernatant to a new tube. The supernatant is the cell 

lysate. Store at -80°C in single-use aliquots. 

For suspension cells 

1. Remove media by low speed centrifugation (~1,200 rpm) when the culture reaches 0.5–1.0 x 10 6 viable cells/ml. Treat cells 

by adding fresh media containing regulator for desired time. 

2. Collect cells by low speed centrifugation (~1,200 rpm) and wash once with 5–10 ml ice-cold 1X PBS. 

3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF. 




C. Test Procedure 

1. After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells 

in the strip holder. Unused microwells must be resealed in the storage bag and stored at 4°C immediately. 

2. Cell lysates can be undiluted or diluted with sample diluent (supplied in each PathScan ® Sandwich ELISA Kit, blue color). 

Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for lysates 

and kit assay results. 

3. Add 100 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of 

microwells. Incubate the plate for 2 hr at 37°C. Alternatively, the plate can be incubated overnight at 4°C. 

4. Gently remove the tape and wash wells: 

a. Discard plate contents into a receptacle. 

b. Wash 4 times with 1X wash buffer, 200 µl each time per well. 

c. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not 

allow wells to completely dry at any time. 

d. Clean the underside of all wells with a lint-free tissue. 

5. Add 100 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at 37°C for 1 hr. 

6. Repeat wash procedure (Section C, Step 4). 

7. Add 100 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate for 30 min at 37°C. 


9. Add 100 µl of TMB substrate to each well. Seal with tape and incubate the plate for 10 min at 37°C or 30 min at 25°C. 

10. Add 100 µl of STOP solution to each well. Shake gently for a few seconds. 

NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP solution. 

11. Read results 

a. Visual Determination: Read within 30 min after adding STOP solution. 

b. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm 

within 30 min after adding STOP solution. 


Phospho cell lysates 

serve as convenient 

and well-validated 

positive controls for 

ELISA studies. 

PathScan ® Sandwich ELISA 

Control Phospho Cell Extracts I 

#7988: ELISA analysis of #7988 using 

multiple PathScan ® Sandwich ELISA 

kits. Samples were prepared using the 

standard ELISA protocol, to a final concentration 

of 0.25 mg/ml and assayed 

using the indicated ELISA kits. 


5 

4.5 

4 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

Phospho-Akt 

(Thr308) 

Total Bad 

Total NF-κB 

p65 

PathScan ® Sandwich ELISA Chemiluminescent Protocol 

NOTE: Refer to product-specific datasheets or product webpage for assay incubation temperature. This chemiluminescent ELISA 

is offered in low volume microplate. Samples and reagents only require 50 µl per microwell. 


For lyophilized formulation 


1. Microwell strips: Bring all to room temperature before use. 

2. Detection Antibody: Supplied lyophilized as a green colored cake or powder. Add 1.0 ml of Detection Antibody Diluent (green 

solution) to yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully 

reconstitute. To make the final working solution, add the full 1.0 ml volume of reconstituted Detection Antibody to 10.0 ml of 

Detection Antibody Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C. 

3. HRP-Linked Antibody: Supplied lyophilized as a red colored cake or powder Add 1.0 ml of HRP Diluent (red solution) to 

yield a concentrated stock solution. Incubate at room temperature for 5 min with occasional gentle mixing to fully reconstitute. 

To make the final working solution, add the full 1.0 ml volume of reconstituted HRP-Linked Antibody to 10.0 ml of HRP 

Diluent in a clean tube and gently mix. Unused working solution may be stored for 4 weeks at 4°C. NOTE: Some PathScan ® 

ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody. 

4. Detection Antibody Diluent: Green colored diluent for reconstitution and dilution of the detection antibody (5.5 ml provided). 

5. HRP Diluent: Red colored diluent for reconstitution and dilution of the HRP‐Linked Antibody (5.5 ml provided). 

6. Sample Diluent: Blue colored diluent provided for dilution of cell lysates. 

7. 1X Wash Buffer: Prepare by diluting 20X Wash Buffer (included in each PathScan ® Sandwich ELISA Kit) in purified water. 





9. Luminol/Enhancer Solution and Stable Peroxide Buffer 

For liquid formulation 



2. Bring all microwell strips to room temperature before use. 

3. Prepare 1X wash buffer by diluting 20X Wash Buffer (included in each PathScan ® Sandwich ELISA Kit) in dH 2 O. 





5. 20X LumiGLO ® Reagent and 20X Peroxide: (#7003) 




desired time. 


3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the 






Total 

S6 RP 

p-S6 

(Ser235/236) 

p-CREB 

(Ser133) 

Total 

CREB 

p-PRAS40 

(Thr246) 

p-RSK1 

(Ser380) 


223


chapter 13: Sandwich eLISA 

Validation of CST 

Cell Lysis Buffer 

for detection of an 

intracellular target 





3. Cells harvested from 50 ml of growth medium can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF. 




C. Test Procedure 

1. After the microwell strips have reached room temperature, break off the required number of microwells. Place the microwells 

in the strip holder. Unused microwells must be resealed in the storage bag and stored at 4°C immediately. 

2. Cell lysates can be used undiluted or diluted with sample diluent (supplied in each PathScan ® Sandwich ELISA Kit, blue 

color). Individual datasheets or product webpage for each kit provide information regarding an appropriate dilution factor for 

lysates and kit assay results. 

3. Add 50 µl of each undiluted or diluted cell lysate to the appropriate well. Seal with tape and press firmly onto top of 

microwells. Incubate the plate for 2 hr at room temperature. Alternatively, the plate can be incubated overnight at 4°C. 

4. Gently remove the tape and wash wells: 


b. Wash 4 times with 1X Wash Buffer, 150 µl each time per well. 

c. For each wash, strike plates on fresh paper towels hard enough to remove the residual solution in each well, but do not 

allow wells to dry completely at any time. 

d. Clean the underside of all wells with a lint-free tissue. 

5. Add 50 µl of detection antibody (green color) to each well. Seal with tape and incubate the plate at room temperature for 1 hr. 


7. Add 50 µl of HRP-linked secondary antibody (red color) to each well. Seal with tape and incubate the plate at room 

temperature for 30 min. 


9. Prepare detection reagent working solution by mixing equal parts 2X LumiGLO ® Reagent and 2X Peroxide. 

10. Add 50 µl of the detection reagent working solution to each well. 

11. Use a plate-based luminometer set at 425 nm to measure Relative Light Units (RLU) within 1–10 min following addition of 

the substrate. 

a. Optimal signal intensity is achieved when read within 10 min. 

RLU (x10 5 ) 

14 

12 

10 

8 

6 

4 

2 

0 

PathScan ® Sandwich ELISA Antibody Pair Protocol 

NOTE: This protocol includes plate preparation 

6 

4 

2 

0 

0.01 0.02 0.03 

0.1 0.2 0.3 0.4 

Protein conc. of lysate (mg/ml) 

0.5 

Cell Lysis Buffer (10X) #9803: The relationship between protein 

concentration of lysates from A-431 cells, untreated or treated with 

Human Epidermal Growth Factor (hEGF) #8916, and immediate light 

generation with chemiluminescent substrate using PathScan ® Total 

EGF Receptor Chemiluminescent Sandwich ELISA Kit #7297. After 

starvation, A-431 cells (85% confluence) were treated with hEGF 

(100 ng/ml, 5 min at 37°C) and then lysed using #9803. Graph 

inset corresponding to the shaded area shows high sensitivity and a 

linear response at the low protein concentration range. 

EGF-treated 

Control 




2. Wash Buffer: 1X PBS/0.05% Tween ® 20, (20X PBST #9809) 

3. Blocking Buffer: 1X PBS/0.05% Tween ® 20, 1% BSA 





5. Bovine Serum Albumin (BSA): (#9998) 



NOTE: Reagents should be made fresh daily. 




desired time. 


3. Remove PBS and add 0.5 ml ice-cold 1X Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm diameter) and incubate the 










3. Cells harvested from 50 ml of growth media can be lysed in 2.0 ml of 1X cell lysis buffer plus 1 mM PMSF. 




C. Coating Procedure 

1. Rinse microplate with 200 µl of dH 2 O, discard liquid. Blot on paper towel to make sure wells are dry. 

2. Dilute capture antibody 1:100 in 1X PBS. For a single 96 well plate, add 100 µl of capture antibody stock to 9.9 ml 1X PBS. 

Mix well and add 100 µl/well. Cover plate and incubate overnight at 4°C (17–20 hr). 

3. After overnight coating, gently uncover plate and wash wells: 


b. Wash 4 times with wash buffer, 200 µl each time per well. For each wash, strike plates on fresh paper towels hard 

enough to remove the residual solution in each well, but do not allow wells to completely dry at any time. 

c. Clean the underside of all wells with a lint-free tissue. 

4. Block plates. Add 150 µl of blocking buffer/well, cover plate, and incubate at 37°C for 2 hr. 

5. After blocking, wash plate (Section C, Step 3). Plate is ready to use. 

D. Test Procedure 

1. Lysates can be used undiluted or diluted in blocking buffer. 100 µl of lysate is added per well. Cover plate and incubate at 

37°C for 2 hr. 

2. Wash plate (Section C, Step 3). 

3. Dilute detection antibody 1:100 in blocking buffer. For a single 96 well plate, add 100 µl of detection antibody Stock to 

9.9 ml of blocking buffer. Mix well and add 100 µl/well. Cover plate and incubate at 37°C for 1 hr. 


5. Secondary antibody, either streptavidin anti-mouse or anti-rabbit-HRP, is diluted 1:1000 in blocking buffer. For a single 

96 well plate, add 10 µl of secondary antibody stock to 9.99 ml of blocking buffer. Mix well and add 100 µl/well. Cover and 

incubate at 37°C for 30 min. 


7. Add 100 µl of TMB substrate per well. Cover and incubate at 37°C for 10 min. 

8. Add 100 µl of STOP solution per well. Shake gently for a few seconds. 

9. Read plate on a microplate reader at absorbance 450 nm. 

a. Visual Determination: Read within 30 min after adding STOP solution. 

b. Spectrophotometric Determination: Wipe underside of wells with a lint-free tissue. Read absorbance at 450 nm 

within 30 min after adding STOP solution. 

CST Technical Support 

When you contact CST for technical support, you can be confident you will be working with a colleague you can trust, 

striving to provide the highest quality products and services to you, our customer. Please see our Technical Support 

resource page online for contact information. www.cellsignal.com/cstsupport 



225

14 


Western Blotting (WB) 

Protein Detection 

Western blotting (WB) or immunoblotting is the most common technique used for monitoring protein 

expression in cells or tissue. WB can be used to measure total or modification-specific protein levels 

qualitatively or quantitatively by chemiluminescent or fluorescent detection. The WB protocol comprises 

multiple steps beginning with preparation of cell lysate and separation of cellular proteins by molecular 

weight on a polyacrylamide gel matrix (SDS-PAGE). The separated proteins are then transferred to a 

nitrocellulose or polyvinylidene fluoride (PVDF) membrane and detected using target-specific antibodies. 

Prior to detection, the membrane is incubated in a blocking solution to prevent nonspecific antibody 

binding. A wide variety of primary antibodies can be used to probe western blots including modificationspecific, 

motif-specific, or total protein antibodies. Proteins can be detected indirectly using an 

unconjugated primary antibody specific to the target of interest coupled with a secondary antibody 

conjugated to either an enzyme (horseradish peroxidase or HRP) for chemiluminescent detection or an 

infrared dye for fluorescent detection. Alternatively, primary antibodies directly conjugated to HRP can 

be used to detect proteins in a one-step protocol. 

The most common challenges presented by WB analysis are weak signal and high background. 

The tips below will help you address the most common factors responsible for these problems. 

WB Tips for Success 

Incubate with primary antibody overnight 

to improve antibody-target binding. 

Overnight incubation of membrane and primary antibody can greatly enhance signal simply by allowing 

more time for antibody-antigen binding. We have found that overnight incubation, with gentle agitation, 

at 4°C results in stronger antibody specific signal. 

Primary antibody incubation overnight at 

4°C yields significantly increased antibody 

binding compared to a 2 hr incubation. 

Phospho-Akt (Ser473) Antibody #9271: WB analysis of extracts from HeLa 

cells, untreated or treated with LY294002 #9901 or Human Insulin-like Growth 

Factor I (hIGF-I) #8917, using #9271 (top) or PKC Antibody #2058 (bottom). 

kDa 

140 

100 

80 

60 

50 

40 

140 

100 

80 

60 

50 

40 

16 hr, 4°C 

2 hr, RT 

Phospho- 

Akt (Ser473) 

PKCδ 

- - + 

- - + LY294002 

- + + - + + hIGF-I 

Dilute secondary antibody in stronger 

blocking agents to reduce nonspecific signal. 

We suggest diluting secondary antibody in 5% nonfat milk in TBST rather than in 5% BSA in TBST. Milk 

offers stronger blocking of nonspecific binding, hence the BSA-based dilution yields significantly higher 

background than the milk-based dilution. 

Diluting secondary antibody in milk yields 

lower background levels because milk is a 

stronger blocking agent. 

Phospho-Stat3 (Tyr705) Antibody #9131: WB analysis of extracts from Jurkat 

cells, untreated or treated with Human Interferon-α1 (hIFN-α1) #8927, using #9131. 

Blots were incubated in Anti-rabbit IgG, HRP-linked Antibody #7074 diluted in either 

5% nonfat milk in TBST or 5% BSA in TBST, as indicated. 

kDa 

200 

140 

100 

80 

60 

50 

40 

Milk 

BSA 

– + – + 

Weak signal of mid-to-high molecular weight proteins. 

chapter 14: Western Blotting (WB) 

Phospho- 

Stat3 

Due to their large size, high molecular weight proteins do not typically transfer as efficiently as smaller 

proteins. Wet transfer often provides better transfer of mid-to-high molecular weight proteins than 

semi-dry or dry transfer methods. Additionally, because methanol acts as a fixative, decreasing the 

methanol concentration in transfer buffer from 20% to 5% and increasing transfer time to 3 hours can 

also improve transfer efficiency of proteins >200 kDa. 

Wet transfer provides better capture of mid-to-high 

molecular weight proteins and a stronger WB signal. 

Phospho-Stat3 (Tyr705) (D3A7) 

XP ® Rabbit mAb #9145: WB 

analysis of extracts from HeLa 

cells, untreated or treated with 

Human Interferon-α1 (hIFN-α1) 

#8927, using #9145 or a 

phospho-Stat3 (Tyr705) antibody 

from a competitor. Blots were 

transferred using either traditional 

wet transfer methods (left) or 

iBlot ® dry transfer system (right). 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

HeLa 

Wet Transfer 

Dry Transfer 

HeLa HeLa HeLa 

hIFN-α1 

Phospho- 

Stat3 (Tyr705) 

Sonicate lysates to increase nuclear protein recovery. 

20 

kDa 

100 

80 

Sonication thoroughly disrupts both cell and nuclear membranes, improving cell lysis and resulting in 

higher protein levels in clarified lysate. This enhanced membrane disruption is especially important 

for nuclear proteins, which may not be completely released through detergent use alone. If you do not 

have access to a probe sonicator, passing samples through a fine gauge needle will also serve to break 

membranes and shear DNA. 

– + 


– + – + – + 

Alternate 

Provider 


Alternate 

Provider 

hIFN-α1 

60 

50 

40 

30 

Lysate sonication is critical to the observation 

of nuclear and chromatin-bound proteins. 

20 

10 

- 

Sonicated 

+ - + sorbitol 

Unsonicated 

Phospho- 

Histone H3 

(Ser10) 

Phospho-Histone H3 (Ser10) Antibody #9701: WB analysis of extracts from CKR/PAEC cells, 

untreated or treated with sorbitol and either sonicated or without sonication, using #9701. 

WB Protocols and Troubleshooting Videos 

For more in-depth help with WB, please see our online WB Protocols and Troubleshooting videos. 

www.cellsignal.com/wbvideo 


www.cellsignal.com/cstwb 

227



WB General Protocol 

For western blots, incubate membrane with diluted primary antibody in either 5% w/v BSA or nonfat dry milk, 1X TBS, 0.1% 

Tween ® 20 at 4°C with gentle shaking, overnight. NOTE: Please refer to primary antibody datasheet or product webpage for 

recommended primary antibody dilution buffer and recommended antibody dilution. 




2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH 2 O, mix. 

3. 1X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer 

by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH 2 O. 

4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X Running Buffer: add 100 ml 10X running buffer to 

900 ml dH 2 O, mix. 

5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml 

methanol + 700 ml dH 2 O, mix. 

6. 10X Tris Buffered Saline with Tween ® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml 

dH 2 O, mix. 

7. Nonfat Dry Milk: (#9999) 

8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and 

mix well. 

9. Wash Buffer: (#9997) 1X TBST 


11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA or 5% nonfat dry milk as indicated on primary antibody 

datasheet; for 20 ml, add 1.0 g BSA or nonfat dry milk to 20 ml 1X TBST and mix well. 

12. Biotinylated Protein Ladder Detection Pack: (#7727) 

13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720) 

14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm 

is generally recommended. 

15. Secondary Antibody Conjugated to HRP: anti-rabbit (#7074); anti-mouse (#7076) 

16. Detection Reagent: LumiGLO ® chemiluminescent reagent and peroxide (#7003) or SignalFire ECL Reagent (#6883) 

B. Protein Blotting 

A general protocol for sample preparation. 

1. Treat cells by adding fresh media containing regulator for desired time. 

2. Aspirate media from cultures; wash cells with 1X PBS; aspirate. 

3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). 

Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice. 

4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). 

5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice. 

6. Microcentrifuge for 5 min. 

7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm). NOTE: Loading of prestained molecular weight markers (#7720, 10 µl/lane) 

to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended. 

8. Electrotransfer to nitrocellulose membrane (#12369). 

C. Membrane Blocking and Antibody Incubations 

NOTE: Volumes are for 10 cm x 10 cm (100 cm 2 ) of membrane; for different sized membranes, adjust volumes accordingly. 

I. Membrane Blocking 

1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature. 

2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. 

3. Wash three times for 5 min each with 15 ml of TBST. 

For HRP Conjugated Primary Antibodies 

1. Incubate membrane and primary antibody (at the appropriate dilution as recommended in the product datasheet) in 10 ml 

primary antibody dilution buffer with gentle agitation overnight at 4°C. 

2. Wash 3 times for 5 min each with 15 ml of TBST. 

3. Incubate with Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000), to detect biotinylated protein markers, in 10 ml 

of blocking buffer with gentle agitation for 1 hr at room temperature. 


5. Proceed with Detection (Section D). 

For Biotinylated Primary Antibodies 




3. Incubate membrane with Streptavidin-HRP (#3999 at the appropriate dilution) in 10 ml of blocking buffer with gentle 

agitation for 1 hr at room temperature. 



Do not add Anti-biotin, HRP-linked Antibody for detection of biotinylated protein markers. There is no need. The Streptavidin-HRP 

will also visualize the biotinylated markers. 

D. Detection of Proteins 

1. Incubate membrane with 10 ml LumiGLO ® (0.5 ml 20X LumiGLO ® #7003, 0.5 ml 20X Peroxide, and 9.0 ml dH 2 O) or 

10 ml SignalFire #6883 (5 ml Reagent A, 5 ml Reagent B) with gentle agitation for 1 min at room temperature. 

2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 

10 sec exposure should indicate the proper exposure time. NOTE: Due to the kinetics of the detection reaction, signal is 

most intense immediately following incubation and declines over the following 2 hr. 

Phosphatase inhibitors help preserve phosphorylation. 

kDa 

140 

100 

80 

60 

50 

40 

60 

50 

A 

0 3 6 20 0 3 6 20 hrs 

B 

Phospho-Akt 

(Ser 473) 

Selection Guide for Chemiluminescent Detection 

Akt 

Protease/Phosphatase Inhibitor Cocktail (100X) #5872: 

WB analysis of extracts from NIH/3T3 cells, serum-starved 

overnight and treated with hPDGF-BB #8912 (100 ng/ml, 5 

min), prepared in lysis buffer in the absence of phosphatase 

inhibitors (A) or with #5872 added (B) and was incubated at 

37˚C for the indicated time following cell lysis, using Phospho- 

Akt (Ser473) (D9E) XP ® Rabbit mAb #4060 (upper) or Akt (pan) 

(C67E7) Rabbit mAb #4691 (lower). 

Reagent Sensitivity Recommended Application 

LumiGLO ® 

Sub-picogram sensitivity for recombinant protein General use detection reagent, 

Reagent #7003 

most versatile option 

SignalFire ECL 

Reagent #6883 

SignalFire Plus ECL 

Reagent #12630 

SignalFire Elite ECL 

Reagent #12757 

Mid-picogram sensitivity for recombinant protein 

Low-picogram sensitivity for recombinant protein, 

with strong signal lasting for many hours 

Femtogram sensitivity for recombinant protein, with 

low nanogram sensitivity for target protein in cell 

extracts 

General use detection reagent, 

most economical option 

Strong and long-lasting signal, useful for 

reading assays performed in large batches 

Extremely intense signal enables detection 

of low abundance endogenous proteins 

II. Primary Antibody Incubation 

Proceed to one of the following specific set of steps depending on the primary antibody used. 

For Unconjugated Primary Antibodies 

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product datasheet) 

in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C. 


3. Incubate membrane with the species appropriate HRP-conjugated secondary antibody (#7074 or #7076 at 1:2000) and 

anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking 

buffer with gentle agitation for 1 hr at room temperature. 









229



WB Fluorescent Protocol 

For western blots, incubate membrane with diluted primary antibody in either 5% w/v BSA or nonfat dry milk, 1X TBS, 0.1% 

Tween ® 20 at 4°C with gentle shaking, overnight. 

NOTE: Two-color western blots require primary antibodies from different species and appropriate secondary antibodies labeled 

with different dyes. Overlap of epitopes may cause interference and should be considered in two color western blots. If the primary 

antibodies require different primary antibody incubation buffers, test each primary individually in both buffers to determine 

the optimal one for the dual-labeling experiment. 



1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2 

O, mix. 

2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X TBS to 900 ml dH 2 O, mix. 


by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Dilute to 1X with dH 2 O. 

4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X Running Buffer: add 100 ml 10X running buffer 900 ml 

dH 2 O, mix. 

5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer 200 ml 

methanol + 700 ml dH 2 O, mix. 

6. 10X Tris Buffered Saline with Tween ® 20 (TBST-10X): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 

900 ml dH 2 

O, mix. 

7. Nonfat Dry Milk: (#9999) 

8. Blocking Buffer: 1X TBS with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBS and mix 

well. Tween ® 20 should not be present in the Blocking Buffer because it is auto-fluorescent and increases non-specific 

background. After the blocking step, Tween ® 20 can be reintroduced to subsequent diluent buffers. 

9. Wash Buffer: 1X TBST 


11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA or 5% nonfat dry milk as indicated on primary antibody 

datasheet; for 20 ml, add 1.0 g BSA or nonfat dry milk to 20 ml 1X TBST and mix well. 

12. Secondary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 

1X TBST and mix well. (Secondary antibodies; anti-rabbit #5151 and #5366; anti-mouse #5257 and #5470) 

13. Prestained Protein Marker, Broad Range (Premixed Format): (#7720) 

14. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes (recommended). 

Pore size 0.2 µm is generally recommended. 

B. Protein Blotting 

A general protocol for sample preparation. 

1. Treat cells by adding fresh media containing regulator for desired time. 

2. Aspirate media from cultures; wash cells with cold 1X PBS; aspirate. 

3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl per plate of 10 cm diameter plate). 

Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice. 

4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity). 

5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice. 

6. Microcentrifuge for 5 min. 

7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm). 

NOTE: Loading of prestained molecular weight markers (#7720, 10 µl/lane) is recommended to verify electrotransfer and to 

determine molecular weights. Prestained markers are autofluorescent at near-infrared wavelengths. 

8. Electrotransfer to nitrocellulose membrane (#12369). 

C. Membrane Blocking and Antibody Incubations 

NOTE: Volumes are for 10 cm x 10 cm (100 cm 2 ) of membrane; for different sized membranes, adjust volumes accordingly. 

1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature. 

2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. CRITICAL STEP: Do not include Tween ® 20 in 

blocking buffer (Section A, Step 8). 





6. Incubate membrane with fluorophore-conjugated secondary antibody (#5470, #5257, #5366, #5151) (1:5000–1:25,000 

dilution of 1 mg/ml stock) in 10 ml of secondary antibody dilution buffer with gentle agitation for 1 hr at room temperature. 


In the absence of protease inhibitors, β-Catenin signal 

fades within 3 hours after harvest. In the presence of 

protease inhibitors, β-Catenin is significantly slowed. 

Protease/Phosphatase Inhibitor Cocktail (100X) #5872: 

WB analysis of extracts from NIH/3T3 cells, prepared in lysis 

buffer in the absence of protease inhibitors (A) or with #5872 

added (B), and incubated at 37ºC for the indicated time points, 

using β-Catenin (D10A8) XP ® Rabbit mAb #8480. In the absence 

of protease inhibitors, β-Catenin signal fades within 3 hr after 

harvest, indicating protein degradation. In the presence of the 

protease inhibitor cocktail, the β-Catenin degradation is slowed 

significantly and signal is still present at 20 hr following harvest. 

Loading Control Antibodies 

It is important to be certain that WB band intensities reflect the experimental treatment and are not 

attributable to variability in preparing or loading cell extracts. Loading control antibodies should detect a 

relatively abundant protein that does not vary significantly under experimental conditions. For example, 

the “housekeeping” enzyme GAPDH is commonly employed as a loading control for short-term signaling 

studies, but it may not be the optimal loading control in samples where metabolic pathways are affected. 

This table will help you select loading controls on the basis of sample type and subunit molecular 

weight. By choosing a control with a subunit molecular weight different than your target protein, you 

have the option of detecting the loading control in the same lane as your target protein. 

kDa 

140 

100 

80 

60 

50 

0 3 6 20 hrs 

β-Catenin 

degradation 

product 

kDa 

Whole Cell / 

Cytoplasmic Nuclear Mitochondrial Cytoskeletal Plasma Membrane 

Serum & 

Extracellular Fluids 

230 Myosin IIb 

135 Cadherin 

124 Vinculin Vinculin 

116 PARP 

100 α-Actinin NaK-ATPase 

90 HSP 90 

89 PARP 

85 CD71 

77 Transferrin 

74 Lamin A/C 

70 HSP 70 

68 Lamin B1 

63 Lamin A/C 

62 HDAC 1 

60 HSP 60 HSP 60 

52 α-Tubulin 

45 MEK1/2 Lamin B1 β-Actin 

38 TBP 

37 GAPDH 

36 PCNA 

35 β-Tubulin 

34 VDAC/Porin 

24 Caveolin-1 

21 Caveolin-1 

19 Cofilin 

17 Histone H3 COX IV 

9 Profilin 1 

5 Profilin 1 

A 

0 3 6 20 

B 

D. Detection of Proteins 

1. Drain membrane of excess TBST and allow to dry. CRITICAL STEP: Membrane must be dry for fluorescent detection. 

2. Scan membrane using an appropriate fluorescent scanner following the manufacturer’s recommendations. 

230 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys. www.cellsignal.com/cstprotocols 

231



WB Troubleshooting Guide 

High Background: 

General background is high or nonspecific bands that appear after a short exposure of blot to film. Be conscious of 

the fact that motif antibodies, post-translational modifications, and splice variants may result in multiple banding. 

Cause 

Solution 

Lysate 

preparation 

Membrane 

Primary antibody dilution 

and/or incubation 

Inadequate 

washing 

Secondary antibody 

dilution and/or incubation 

Detection reagent 

Exposure time 

Freshly prepared samples result in fewer nonspecific and degradation bands, and therefore 

yield a cleaner blot. In general, tissue extracts tend to contain more background bands and 

degradation products than cell line extracts due to connective tissue. Using fresh, sonicated 

and clarified tissue extracts may lessen background. Samples should always be lysed in 

appropriate buffers that include protease inhibitors and phosphatase inhibitors for phosphotargets 

(Phosphatase Inhibitor Cocktail (100X) #5870, Protease/Phosphatase Inhibitor Cocktail 

(100X) #5872, Protease Inhibitor Cocktail #5871). We recommend the use of Cell Lysis Buffer 

(#9803) or RIPA Buffer (#9806), which contains stronger detergent, for sample preparation 

and protein concentration quantification. SDS loading buffer (#7722 or #7723) may be 

used for whole cell lysis without protein quantification. Chaps Cell Extract Buffer (#9852) is 

used to prepare cytoplasmic cell fractions and is useful for studying caspase signaling. Cell 

Fractionation Kit (#9038) is used to prepare cytoplasmic, membrane/organelle, and nuclear/ 

cytoskeletal cell fractions. 

Use high quality nitrocellulose membrane (Nitrocellulose Sandwiches #12369). Pore size 

0.2 µm is generally recommended; membranes with a pore size of 0.45 µm are not recommended 

for proteins smaller than 30 kDa. PVDF membranes may yield higher background 

than nitrocellulose. Nylon membranes are not recommended for western blotting. Block for 

1 hr at room temperature in 5% nonfat dry milk in TBST. 

Incubate primary antibody overnight at 4°C in TBST at the recommended dilution with the 

recommended blocking agent (either 5% BSA or 5% nonfat dry milk). For individual antibodies 

please consult the product datasheet for recommended dilution buffer. 

Washing for less time than the recommended 3 times 5 min in TBST is common, and can 

result in high background. We recommend that washes be timed to ensure accuracy. Additionally, 

low Tween ® 20 can contribute to high background; we recommend 0.1% Tween ® 20. 

This applies to washing steps after both primary and secondary antibody incubations. 

Some secondary antibodies bind nonspecifically to proteins in cell extracts. To assess the 

quality of a secondary antibody, perform a blot (through to film exposure) without primary antibody. 

Serial dilutions of the secondary antibody can be performed on blots with the same cell 

extracts and primary antibody to optimize secondary antibody concentration. Always incubate 

the secondary antibody in 5% nonfat dry milk in TBST for 1 hr at room temperature, diluting 

the secondary antibody in BSA yields higher levels of background bands. CST secondary 

antibodies have already been optimized and do not require titration. 

Chemiluminescent detection requires that high purity water be used to dilute concentrated 

reagents. Use only purified water with organic and inorganic impurities removed. We recommend 

using RODI water. Additionally, ensure that the membrane is never allowed to dry out 

during the antibody incubation process prior to detection reagent exposure. 

Film exposure times of more than 30 sec lead to increased background signal. To avoid long exposure 

times, it is important to use cell lines or tissues with adequate protein expression levels 

and use recommended primary antibody incubation conditions (see primary antibody dilution 

and/or incubation section). If necessary, use treatment to induce expression or modification. 

Low Signal: 

The protein of interest cannot be detected after a short exposure of blot to film. 

Cause 

Solution 

Lysate preparation 20–30 µg total protein from whole cell extracts per lane is usually sufficient for detection. If 

basal levels of target protein, or protein modification are low, it may be necessary to induce 

expression or modification via chemical stimulant. You may want to investigate alternative cell 

lines or tissues in which the protein of interest is more abundant. Samples should always be 

lysed in appropriate buffers that include protease inhibitors and phosphatase inhibitors, for 

phospho-targets (Phosphatase Inhibitor Cocktail (100X) #5870, Protease/Phosphatase Inhibitor 

Cocktail (100X) #5872, Protease Cocktail #5871). Lysates should always be sonicated to 

ensure efficient protein extraction of chromatin and membrane-bound targets. Please visit the 

Controls Table on our website for suggested positive controls and treatments. 

Primary antibody dilution 

and/or incubation 

SDS-PAGE Gel selection 

Transfer and Membrane 

Blocking 

Washing 

Secondary antibody 

dilution and/or incubation 

Incubate primary antibody overnight at 4°C in TBST at the recommended dilution with the 

recommended blocking agent (5% BSA or 5% nonfat dry milk). The use of alternate blocking 

agents, such as gelatin, serum, protein-free blocking agents, casein, or mixed blocking agents 

may reduce target signal intensily. For optimal results with individual antibodies, please consult 

the product datasheet for recommended dilution buffer. 

In general, Tris-Glycine gels are recommended. However, for high molecular weight proteins 

we recommend Tris-Acetate gels and associated buffers. Proteins transfer more efficiently 

from 1 mm gels than from 1.5 mm gels. The use of thicker gels can result in incomplete 

transfer of high molecular weight proteins, so we recommend monitoring transfer efficiency 

and implementing modifications of transfer conditions to optimize for the size of your protein 

target of interest. 

Incomplete transfer can be corrected by longer transfer or by the use of higher voltage. In general, 

we recommend including 20% methanol in transfer buffer, and performing a wet transfer 

for 1.5 hr at 70 V. For high molecular weight proteins, we recommend reducing the methanol 

in transfer buffer to 5% to improve transfer efficiency and avoid fixing large proteins in the 

gel matrix, as well as increasing transfer time to 3 hr at 70 V. Over transfer (protein transfer 

through membrane) can be problematic when examining smaller proteins. For small proteins, 

it is important to use a 0.2 µm pore size membrane and wet transfer for 1.5 hr at 70 V, not a 

0.45 µm pore size membrane or transferring overnight, which may result in over transfer and 

a lack of small protein signal. 

Blocking the membrane for too long can obscure antigenic epitopes and prevent the antibody 

from binding. Block for only 1 hr at room temperature. 

Washing for longer than the recommended 3 times 5 min is a common issue and can result 

in reduced signal. We recommend that washes be timed to ensure accuracy. TBST should 

contain 0.1% Tween ® 20. This applies to washing steps after both primary and secondary 

antibody incubations. Additionally, we recommend washing in TBST, as washing in PBST has 

been shown to result in reduced signal. 

Serial dilutions of the secondary antibody can be performed on blots with the same cell 

extracts and primary antibody to optimize secondary antibody concentration. Always incubate 

the secondary antibody in 5% nonfat dry milk in TBST for 1 hr at room temperature. Avoid 

including sodium azide in HRP-conjugated secondary antibody buffers as azide inhibits HRP 

activity. CST secondary antibodies have already been optimized and do not require titration. 

Detection reagent 

Use biotinylated molecular weight standards that can be detected with anti-biotin-HRP as 

positive controls for chemiluminescent detection. 

WB detection of 

subcellular protein 

markers verifies 

effectiveness of 

detergent-based 

cell fractionation. 

kDa 

100 

80 

60 

50 

40 

30 

20 

10 

A B C D 

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 

Cell Fractions 

1. Whole cell lysates 

2. Cytoplasmic 

3. Membrane 

4. Nuclear/Cytoskeletal 

Cell Fractionation Kit #9038: WB analysis of HeLa cell fractions were prepared using #9038. Subcellular localization was determined 

using antibodies from the Cell Fractionation Antibody Sampler Kit #11843 [MEK1/2 (D1A5) Rabbit mAb #8727 (A), AIF (D39D2) XP ® Rabbit 

mAb #5318 (B), Histone H3 (D1H2) XP ® Rabbit mAb #4499 (C), and Vimentin (D21H3) XP ® Rabbit mAb #5741 (D)]. 







233

15 


For targets that run 

near 50 kDa, use 

light chain-specific or 

conformation-specific 

secondary antibody to 

avoid obscuring the 

intended target band. 

Immunoprecipitation (IP) 

Protein Enrichment 

Immunoprecipitation (IP) uses antibodies bound to protein A or G beads to isolate target proteins from 

a cell lysate. IP is often used as an enrichment strategy to study low abundance proteins that cannot 

be detected directly by western blot. IP is also used to identify members of protein complexes, as 

proteins associated with the target protein will co-precipitate with the antibody-bound beads. Proteins 

are either eluted off the beads prior to western blot, or the bead-bound protein can be directly analyzed 

by western blot. 

The stringency of IP buffers is key for experimental success. High stringency buffers that contain 

anionic detergents such as SDS may work for IP but are not recommended for co-IP experiments, as 

they dissociate protein complexes. Medium and low stringency buffers that contain nonionic detergents 

such as NP-40 or Triton X-100 are better suited for co-IP experiments because they allow protein 

associations to remain intact. Successful IP and co-IP experiments require specific and sensitive antibodies 

to minimize enrichment of non-specific proteins. While unconjugated antibodies are commonly 

used for IP experiments, alternative detection options include biotinylated primary antibodies combined 

with streptavidin beads or primary antibodies directly conjugated to IP beads. IP beads are available in 

agarose, Sepharose ® , or magnetic-bead formats. Agarose and Sepharose beads have a higher protein 

binding capacity, but require multiple centrifugation steps, which increases experimental time and 

can lead to loss of beads due to pipetting. Magnetic beads are a convenient option as the beads are 

precipitated using a magnetic rack, thereby eliminating several centrifugation steps. 

A common challenge presented by IP analysis is the presence of an IgG chain band obscuring the 

western blot band of interest. The tips below offer options to clarify your results by avoiding crossreacting 

IgG bands. 

IP Tips for Success 

Use conformation-specific secondary antibodies for WB analysis 

of an IP if the target size is similar to an IgG subunit size. 

The antibody used for IP remains in the sample and often appears on the subsequent western blot as 

bands at 50 kDa (IgG heavy chain) and 25 kDa (IgG light chain). The conformation-specific secondary 

antibody does not recognize denatured heavy or light IgG chains, so the 25 and 50 kDa bands do not 

appear on the western blot. The light chain-specific secondary antibody only recognizes the IgG light 

chain, so the 50 kDa heavy chain does not appear on the blot. 

kDa 

140 

100 

80 

60 

50 

40 

30 

20 

A B C 

1 2 1 2 1 2 

Heavy 

Chain 

PRAS40 

Where possible, use primary antibodies 

from different species for the IP and the WB. 

IgG chains from the IP antibody are recognized by the secondary antibody when the western blot 

primary antibody is raised in the same host species (For example, the IgG chains will be detected by 

the anti-rabbit secondary antibody when rabbit antibodies are used for IP and western blot). Using IP 

and western blot antibodies raised in different species avoids secondary antibody recognition of the IgG 

heavy chain and light chain from the IP. 


Light 

Chain 

Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb 

#3677: IP of PRAS40 from untreated HeLa cells using PRAS40 (D23C7) 

Rabbit mAb #2691. Secondary antibodies include Mouse Anti-rabbit 

IgG (Conformation Specific) (L27A9) mAb #3678 (A), #3677 (B), and 

Anti-rabbit IgG, HRP-linked Antibody #7074 (C). The bound Mouse 

Anti-Rabbit IgG mAb was detected by Anti-mouse IgG, HRP-linked 

Antibody #7076 (A,B). The positions of the reduced and denatured 

rabbit IgG heavy and light chains are indicated. 

Lanes 

1. 10% input of untreated HeLa cells 

2. IP of PRAS40 from untreated HeLa cells using PRAS40 (D23C7) 


Akt (pan) (C67E7) Rabbit mAb #4691 and Akt 

(pan) (40D4) Mouse mAb #2920: IP of Akt from 293 

cells treated with Human Insulin-like Growth Factor I 

(hIGF-I) #8917. WB analysis performed with #4691 and 

Anti-rabbit IgG, HRP-linked Antibody #7074 (A) or with 

#2920 and Anti-mouse IgG, HRP-linked Antibody #7076 

(B). Note in lane 5 that the rabbit IgG heavy chain is 

recognized by #7074 (A), but not by #7076 (B). 

Lanes 

1. Beads and 293 + hIGF-I lysate control 

2. Rabbit IgG control 

3. Mouse IgG control 

4. 10% input of 293 + hIGF-I lysate (no IP) 

5. IP: Akt (pan) (C67E7) Rabbit mAb #4691 

kDa 

140 

100 

80 

60 

50 

40 

30 

IP Native Protein Protocol 

This protocol is intended for IP of native proteins for analysis by western immunoblotting or kinase activity. 

20 

A 

B 

1 2 3 4 5 1 2 3 4 5 



1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH 2 

O, mix. 

2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH 2 

O, mix. 

NOTE: Add 1 mM PMSF (#8553) immediately prior to use. 


by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. 

4. Protein A or G Agarose Beads (For unconjugated primary antibodies): Use Protein A (#9863, #8687) for rabbit IgG IP 

and Protein G (#8740) for mouse IgG IP. NOTE: Magnetic beads (#8687, #8740) require 6-Tube Magnetic Separation Rack 

(#7017). 

5. Immobilized Streptavidin (Bead Conjugate) (For biotinylated antibodies): (#3419) Gently vortex vial and use 10 µl per IP. 

6. 10X Kinase Buffer (for kinase assays): (#9802) To prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 

900 µl dH 2 O, mix. 

7. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X 

kinase buffer. 


1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time. 

2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS. 

3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min. 

4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice. 

5. Sonicate on ice 3 times for 5 sec each. 

6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. 

If necessary, lysate can be stored at -80°C. 

C. Immunoprecipitation 

Cell Lysate Pre-Clearing (Optional step for unconjugated and biotinylated antibodies.) 

1. Add 10–30 µl of 50% bead slurry, either Protein A or G agarose or magnetic beads (for unconjugated primary antibodies) or 

10 µl streptavidin beads (#3419; for biotinylated antibodies), to 200 µl cell lysate at 1 mg/ml. 

2. Incubate with rotation at 4°C for 30–60 min. 

3. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube. 

4. Proceed to one of the following specific set of steps depending on the primary antibody used. 

Using Unconjugated Primary Antibodies 

1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate at 1 mg/ml. 

Incubate with gentle rocking overnight at 4°C. 

2. Add either protein A or G agarose or magnetic beads (10–30 µl of 50% bead slurry). Incubate with gentle rocking for 1–3 hr 

at 4°C for agarose beads, or 10–30 min for magnetic beads. 

3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice between washes. 

4. Proceed to Analyze by Western Immunoblotting or Analyze by Kinase Assay (Section D). 

Using Biotinylated Primary Antibodies 

1. Add biotinylated antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate 

at 1 mg/ml. Incubate with gentle rocking overnight at 4°C. 

2. Gently mix Immobilized Streptavidin (Sepharose ® Bead Conjugate #3419) and add 10 µl of slurry. Incubate with gentle 

rocking for 2 hr at 4°C. 

3. Microcentrifuge for 30 sec at 4°C. Wash pellet 5 times with 500 µl of 1X cell lysis buffer. Keep on ice during washes. 

4. Proceed to Sample Analysis (Section D). 

chapter 15: Immunoprecipitation (IP) 

Akt 

IgG 

heavy 

chain 

IgG 

light 

chain 

Use a mouse derived 

western blot antibody 

to avoid recognition 

of IP antibody rabbit 

IgG chains. 

Use magnetic beads 

to reduce protocol 

time by skipping 

centrifugation steps. 

kDa 

140 

100 

80 

60 

50 

40 

30 

20 

1 2 

HC 

Syntaxin 6 

Protein A Magnetic Beads #8687: 

IP of Syntaxin 6 from COS-7 cells using 

Syntaxin 6 (C34B2) Rabbit mAb #2869 

and #8687. WB analysis was performed 

using Syntaxin 6 (C34B2) Rabbit mAb 

#2869. Magnetic beads shorten the IP 

protocol by eliminating centrifugation 

steps after washing and also reduce loss 

of beads due to pipetting. 

Lanes 

1. IP pellet 

2. IP supernatant 

www.cellsignal.com/cstip 

235


Using Immobilized Antibodies (Sepharose ® Bead Conjugate) 

1. Vortex gently and add immobilized bead conjugate (10 µl) to 200 µl cell lysate at 1 mg/ml. Incubate with gentle rocking 

overnight at 4°C. 

2. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes. 


Using Immobilized Antibodies (Magnetic Bead Conjugate) 

1. Vortex gently and add immobilized bead conjugate (10 µl) to 200 µl cell lysate at 1 mg/ml. Incubate with gentle rocking 

overnight at 4°C. 

2. Pellet magnetic beads by placing the tubes in a magnetic separation rack (#7017) and wait 1–2 min for solution to clear. 

Wash pellet five times with 500 µl of 1X cell lysis buffer. Keep on ice during washes. 


D. Sample Analysis 

Proceed to one of the following specific set of steps. NOTE: For magnetic beads, do not centrifuge. Instead use a magnetic 

separation rack (#7017). 

For Analysis by Western Immunoblotting 

1. Resuspend the pellet with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec. 

2. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g. 

3. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE. 

4. Analyze sample by WB General Protocol (Section B, Step 7). 

NOTE: For proteins with molecular weights near 50 kDa, we recommend using Mouse Anti-rabbit IgG (Light-Chain Specific) 

(L57A3) mAb #3677 or Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 as a secondary antibody to 

minimize masking produced by denatured heavy chains. For proteins with molecular weights near 25 kDa, Mouse Anti-rabbit 

IgG (Conformation Specific) (L27A9) mAb #3678 or Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) 

#5127 is recommended. 

For Analysis by Kinase Assay 

1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice. 

2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate. 

3. Incubate for 30 min at 30°C. 

4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec. 

5. Transfer supernatant containing phosphorylated substrate to another tube. 


7. Load the sample (15–30 µl) on SDS-PAGE (4–20%). 

IP Denatured Protein Protocol 

This protocol is intended for immunoprecipitation of denatured proteins where the epitope of interest may not be accessible in 

the native conformation. 


NOTE: Prepare solutions with reverse osmosis or equivalently purified water (dH 2 O). 

1. Denaturing Cell Lysis Buffer: 50 mM Tris (pH 7.5), 70 mM β-Mercaptoethanol (β-ME) Add β-ME just prior to use. Pre-boil 

for 10 min. 

2. 10X Cell Lysis Buffer: (#9803) 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM 

sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na 3 VO 4 , 1 µg/ml Leupeptin 

To prepare 10 ml of 1X Cell Lysis Buffer, add 1 ml of 10XCell Lysis Buffer to 9 ml dH 2 O, mix. NOTE: Add 1 mM PMSF (#8553) 

immediately prior to use. 

3. Protein A or G Agarose Beads (For unconjugated primary antibodies): Use Protein A (#9863, #8687) for rabbit IgG IP 

and Protein G (#8740) for mouse IgG IP. 

4. 3X SDS Loading Buffer: (#7722) 187.5 mM Tris-HCl (pH 6.8 at 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% 

w/v bromophenol blue 


1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time. 

2. To harvest cells under denaturing conditions, remove media and rinse cells once with 1X PBS. 

3. Remove 1X PBS and add 0.4 ml Denaturing Cell Lysis Buffer (preboiled for 10 min immediately prior to use) to each plate 

(150 x 25 mm). 

4. Immediately scrape cells off the plate and transfer to 2.5 ml tubes. 

5. Boil for 10 min. 

6. Add 4 volumes (1.6 ml) 1X ice-cold Cell Lysis Buffer. This is the cell lysate. If necessary, lysate can be stored at –80°C. 

C. Immunoprecipitation 

1. Add primary antibody to 200 µl cell lysate; incubate with gentle rocking overnight at 4°C. 

2. Add Protein A Agarose Beads (20 µl of 50% bead slurry). Incubate with gentle rocking for 1–3 hr at 4°C. 

3. Wash pellet 5 times with 500 µl of 1X Cell Lysis Buffer. microcentrifuge for 30 sec at 4°C. Keep on ice during washes. 

4. Resuspend the pellet with 20 µl 3X SDS Loading Buffer. Vortex, then microcentrifuge for 30 sec. 


6. Load the sample (15–30 µl) on a 4–20% gel for SDS-PAGE. 

7. Analyze sample by WB General Protocol (Section B, Step 7). 

chapter 15: Immunoprecipitation (IP) 

Use IgG isotype controls to estimate the 

nonspecific binding of primary antibodies. 

Rabbit (DA1E) mAb IgG XP ® Isotype Control #3900: IP of XAF1 from Jurkat cells 

treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml, overnight) using #3900 

(lane 2) or #13805 (lane 3). WB analysis was performed using XAF1 (E1E4O) Rabbit mAb 

#13805. In this example, there is minimal nonspecific binding of primary antibodies. 

Lanes 

1. 10% Lysate input 

2. Rabbit (DA1E) mAb IgG XP ® Isotype Control #3900 

3. XAF1 (E1E4O) Rabbit mAb #13805 

kDa 

140 

100 

80 

60 

50 

40 

30 

1 2 3 

IgG heavy 

chain 

XAF1 

20 

– + – 

– – + 

Rabbit (DA1E) mAb IgG 

XP ® Isotype Control 

XAF1 (E1E4O) Rabbit mAb 







237

16 


chapter 

Control Treatments and Cell Lines 

This table displays cell lines, treatments, and CST Control Cell Extracts that can be used as positive 

controls for activation-state specific antibodies. The information found in this table should be seen as a 

starting point for troubleshooting. 

Autophagy 

LC3A 

LC3B 

Cell Line Treatment Control 

HeLa, KNRK, 

NIH/3T3 

HeLa, KNRK, 

NIH/3T3 

16: Control Treatments and Cell Lines 

Chloroquine (50 μM for 20 hr) #11972 

Chloroquine (50 μM for 20 hr) #11972 

Phospho-ULK1 (Ser555) MCF7 Oligomycin (0.5 µM for 30 min) 

To demonstrate phospho-specificity, 

cell extracts or nitrocellulose 

membranes can be subjected to 

phosphatase treatment. 

All UV treatments were carried out 

using a Stratagene Stratalinker ® UV 

Crosslinker. 

Where “transfected” is indicated 

in the cell line column, transfected 

levels of the respective target 

protein were detected. 

Control 

Treatments 

by Target 

Please visit our website for the 

most up-to-date listing of control 

treatments and cell lines. 

www.cellsignal.com/cstcontrols 


Adhesion and Extracellular Matrix 

Phospho-Afadin (Ser1718) A-431 Serum-starve overnight, 

hEGF (100 ng/ml for 5 min) 

Phospho-Catenin δ-1 (Tyr228/Tyr904) A-431 Serum-starve overnight, 


Phospho-Connexin 43 (Ser368) COS-7 Serum-starve overnight, 

TPA (200 nM for 30 min) 

Phospho-FAK (Tyr397/Tyr576/Tyr577/Tyr925) A549 Untreated 

Phospho-p130 Cas (Tyr165/Tyr249/Tyr410) NIH/3T3 Untreated 

Phospho-Paxillin (Tyr118) 

C2C12, A-431, 

C6 NIH/3T3 

Untreated 

Apoptosis 

Cleaved Caspase-1 (Asp297) THP-1 TPA (80 nM overnight) plus 

LPS (1 µg/ml for 8 hr) 

Cleaved Caspase-3 (Asp175) HeLa Serum-starve overnight, 

#9663 

Staurosporine (1 μM for 3 hr) 


#9663 



#9663 


Cleaved Caspase-8 (Asp391/Asp384/Asp387) Jurkat Etoposide (25 μM for 5 hr) #2043 

Cleaved Caspase-9 (Asp315/Asp330) Jurkat Etoposide (25 μM for 5 hr) #2043 

Cleaved DFF45 (Asp224) Jurkat Etoposide (25 µM for 5 hr) #2043 

Cleaved Lamin A (Asp230) HeLa Serum-starve overnight, 

#9663 


Cleaved Lamin A (Small Subunit) HeLa Serum-starve overnight, 

#9663 


Cleaved PARP (Asp214) HeLa Serum-starve overnight, 

#2043 

Staurosporine (1 µM for 3 hr) 

Cleaved α-Fodrin (Asp1185) HeLa Serum-starve overnight, 

#9663 


Phospho-AP2M1 (Thr156) HeLa H 2 O 2 (4 mM for 30 min) #9663 

Phospho-Bad (Ser112/Ser136/Ser155) COS-7 Serum-starve overnight, 


Phospho-Bcl-2 (Ser70) Jurkat Paclitaxel (1 μM for 20 hr) 

Phospho-Bcl-2 (Thr56) RL-7 Nocadazole (1 μg/ml for 20 hr) 

Phospho-Bim (Ser55/Ser69) A20 Serum-starve overnight, 


Phospho-c-Myc (Thr58/Ser62) A-431 Serum-starve overnight, 


Phospho-Caspase-9 (Thr125) MCF7 hEGF (10 ng/ml for 30 min) 

Phospho-FADD (Ser194) Jurkat Inhibit with hydroxyurea 

(4.0 mM for 24 hr); activate with 

Nocodazole (1.0 μg/ml) for 24 hr 

Phospho-Lamin A/C (Ser22) THP-1 Nocadazole (1 μg/ml for 20 hr) 

Phospho-Mcl-1 (Ser159/Thr163) H929 MG132 (10 μM for 2 hr) 

Phospho-Mst1 (Thr183)/Mst2 (Thr180) WEHI Staurosporine (1 µM for 30 min–3 hr) 

Phospho-PAR-4 (Thr163) HeLa Calyculin A (100 nM for 5 min) 

Phospho-PEA-15 (Ser104) C6 TPA (200 nM for 1 hr) 


#9293 

Calcium, cAMP and Lipid Signaling 

Phospho-cPLA2 (Ser505) HeLa Serum-starve overnight, 


Phospho-IP3 Receptor (Ser1756) 

mouse & rat 

brains 

Phospho-MARCKS (Ser152/Ser156) HeLa, NIH/3T3 Serum-starve overnight, 


Phospho-PKA C (Thr197) HeLa Untreated 

Phospho-PKC (pan) HeLa, NIH/3T3 Serum-starve overnight, 


Phospho-PKD/PKCμ (Ser744/Ser748/Ser916) HeLa, NIH/3T3 Serum-starve overnight, 


Phospho-PKM2 (Tyr105) A549 Untreated 

Phospho-PLCβ3 (Ser537/Ser1105) HeLa Serum-starve overnight, 


Phospho-PLCγ1 (Tyr783/Ser1248) NIH/3T3 Serum-starve overnight, 

PDGF (50 ng/ml for 30 min) 

Phospho-PLCγ2 (Tyr759/Tyr1217) Ramos, Raji IgM (12 µg/ml for 2 min) 

Phospho-PLD1 (Thr147/Ser561) HeLa Serum-starve overnight, 


Phospho-PRK1 (Thr774)/PRK2 (Thr816) HeLa Untreated 

Cell Cycle/Checkpoint Control 

Acetyl-p53 (Lys379/Lys382) MCF7 Doxorubicin (0.5 µM for 24 hr); 

400 nM TSA can be used in conjunction 

to stabilize activated protein 

Phospho-53BP1 (Ser25/Ser29/Thr543/Ser1778) HT-29 UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-ATM (Ser1981) 293 IR (10 Gy), 1 hr recovery 

Phospho-ATR (Ser428) HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-ATRIP (Ser224) HeLa Untreated 

Phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora HT-29 Nocodazole (100 ng/ml for 18 hr) 

C (Thr198) 

Phospho-BRCA1 (Ser1524) HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-cdc2 (Thr14/Tyr161) HeLa Nocodazole (100 ng/ml for 18 hr) 

Phospho-cdc2 (Thr15) HeLa Untreated 

Phospho-cdc25C (Thr48/Ser216) HT-29 Nocodazole (100 ng/ml for 18 hr) 

Phospho-CDK2 (Thr160) HeLa Hydroxyurea (10 mM for 16 hr) 

Phospho-CDK9 (Thr186) HeLa Untreated 

Phospho-Chk1 (Ser280/Ser296/Ser317/Ser345) HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-Chk2 (Ser19/Ser33/Ser35/Thr68/Thr387/ HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Thr432/Ser516) 

Phospho-Cyclin B1 (Ser133/Ser147) HeLa Nocodazole (100 ng/ml for 18 hr) 

Phospho-Cyclin D1 (Thr286) HT-1080 MG132 (10 µM for 4 hr) 

Phospho-Cyclin E (Thr62) MCF7 Hydroxyurea (10 mM for 8 hr) 

Phospho-FANCD2 (Ser222) HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-LATS1 (Ser909) HeLa TPA (200 nM for 30 min) 

Phospho-LATS1 (Thr1079) HeLa Okadaic Acid (1 μM for 1 hr) 

Phospho-MDM2 (Ser166) MCF7 hIGF-I (100 ng/ml for 15 min) 

Phospho-Mre11 (Ser676) HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-Myt1 (Ser83) HeLa Nocodazole (100 ng/ml for 18 hr) 

#9160 

#9160 

#9160 

#9160 

#9160 

#9160 


239


chapter 16: Control Treatments and Cell Lines 

Control 

Treatments 

by Target 






Phospho-NPM (Ser4/Thr95/Thr199) HeLa Nocodazole (100 ng/ml for 18 hr) 

Phospho-NuMA (Ser395) M059K UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-p53 (Ser33/Thr81/Ser315) HeLa Nocodazole (100 ng/ml for 18 hr) 

Phospho-p53 (Ser392) 293 Hydroxyurea (20 mM for 30 hr) 

Phospho-p53 (Ser6/Ser9/Ser15/Thr18 

293 UV (100 mJ/cm 2 ), 1 hr recovery #9253 

/Ser20/Ser37/Ser46/Ser315) 

Phospho-p57 Kip2 (Thr310) HeLa Dexamethasone (50 nM for 16 hr) 

Phospho-p63 (Ser160/Ser162) ME-180 Nocodazole (100 ng/ml for 18 hr) 

Phospho-p73 (Tyr99) HT-1376 Pervanadate (1 mM for 20 min) 

Phospho-p95/NBS1 (Ser343) HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-PBK/TOPK (Thr9) HT-29 Nocodazole (100 ng/ml for 18 hr) 

Phospho-Pin1 (Ser16) OVCAR8 Forskolin (30 µM for 30 min) 

Phospho-PNK1 (Ser114/Thr118) HeLa UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-PP1α (Thr320) 

HeLa, COS-7, Untreated 

NIH/3T3, C6 

Phospho-Rad17 (Ser645) COS-7 UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-Rb (Ser608/Ser780/Ser795/Ser807/Ser811) HT-29 Nocodazole (100 ng/ml for 18 hr); 

treatment induces slight phosphoshift 

Phospho-RCC1 (Ser11) HT-29 Thymidine (2 mM for 16 hr) followed 

by Nocodazole (100 ng/ml for 18 hr) 

Phospho-Rpb1 CTD (Ser2/5) MCF7 Doxorubicin (0.5 µM for 30 hr) 

Phospho-SMC1 (Ser360/Ser957) 293 UV (100 mJ/cm 2 ), 1 hr recovery 

Phospho-TACC3 (Ser558) HT-29 Thymidine (2 mM for 16 hr), 

add fresh media 

Phospho-TIF1β (Ser824) HeLa IR (10 Gy), 1 hr recovery 

Phospho-TLK1 (Ser695) SK-N-MC Serum-starve overnight 

Phospho-Wee1 (Ser642) A-431 Serum-starve overnight, 

hEGF (100 nM for 5 min) 

Chromatin/Epigenetic Regulation 

Acetyl- and Phospho-Histone H3 (Lys9/Ser10) NIH/3T3 Serum-starve plus TSA (400 nM 

overnight), add 20% serum for 

15 min, then add Calyculin A 

(100 nM for 15 min) 

Acetyl-CBP (Lys1535)/p300 (Lys1499) NIH/3T3 TSA (400 nM overnight) 

Acetyl-Histone H2A (Lys5) NIH/3T3 TSA (400 nM overnight) 

Acetyl-Histone H2B (Lys5/Lys12/Lys20) NIH/3T3 TSA (400 nM overnight) 

Acetyl-Histone H3 (Lys9/Lys14/Lys18/Lys23) NIH/3T3 TSA (400 nM overnight) 

Acetyl-Histone H4 (Lys5/Lys8/Lys12) NIH/3T3 TSA (400 nM overnight) 

Methyl-Histone H3 (Arg2) MCF7 Untreated 

Mono/Di/Tri-Methyl-Histone H3 

NIH/3T3 Untreated 

(Lys4/Lys27/Lys9/Lys36/Lys79) 

Mono/Di/Tri-Methyl-Histone H4 (Lys20) NIH/3T3 Untreated 

Phospho-CTDSPL2 (Ser104) HeLa Untreated 

Phospho-DBC1 (Thr454) 293 UV (100 mJ/cm 2 ), 4 hr recovery 

Phospho-HDAC3 (Ser424) NIH/3T3 Untreated 

Phospho-HDAC4 (Ser246/Ser632)/HDAC5 (Ser259/ HeLa Serum-starve overnight, Pervanadate 

Ser498)/HDAC7 (Ser155/Ser486) 

Phospho-Histone H2A.X (Ser139/Tyr142) 293 UV (40 mJ/cm 2 ), 2 hr recovery 

Phospho-Histone H3 (Thr3/Ser10/Thr11/Ser28) NIH/3T3 Serum-starve overnight, add 20% 

serum for 15 min, then Calyculin A 

(100 nM ) 15 min 

Phospho-HP1γ (Ser83) HeLa IBMX (30 µM) plus Forskolin 

(500 µM) for 30 min 

Phospho-SATB1 (Ser47) Jurkat Untreated 

Phospho-SirT1 (Ser27/Ser47) 293 Untreated 


Cytoskeletal Regulation 

Acetyl-α-Tubulin (Lys40) HeLa TSA (400 nM for 16 hr) 

Phospho-Caveolin-1 (Tyr14) HeLa Serum-starve overnight, H 2 O 2 

(500 μM for 15 min) 

Phospho-Cofilin (Ser3) 

C2C12, Untreated 

NIH/3T3, HeLa 

Phospho-Cortactin (Tyr421) HeLa Serum-starve overnight, H 2 O 2 


Phospho-CrkII (Tyr221) K-562 Untreated 

Phospho-DRP1 (Ser616) HeLa Nocadazole (100 ng/ml for 16 hr) 

Phospho-DRP1 (Ser637) PC-12 Serum-starve overnight, 

Forskolin (20 µM for 1 hr) 

Phospho-Ezrin (Tyr353) A-431 Serum-starve overnight, 


Phospho-Ezrin (Tyr567)/Radixin (Thr564) 

/Moesin (Thr558) 

C2C12, 

NIH/3T3, HeLa 

Untreated 

Phospho-Filamin A (Ser2152) 293 Untreated 

Phospho-MARK Family (Activation Loop) Raji, BaF3 Untreated 

Phospho-Myosin IIa (Ser1943) 

HeLa, A-431, Untreated 

293T 

Phospho-Myosin Light Chain 2 (Thr18/Ser19) C2C12 Untreated 

Phospho-MYPT1 (Ser507/Ser668/Thr853) HeLa Serum-starve overnight, 

Calyculin A (100 nM for 30 min) 

Phospho-Na, K-ATPase a1 (Ser16/Ser23) PC-12 Serum-starve overnight, 


Phospho-Na, K-ATPase a1 (Tyr10) A-431 Serum-starve overnight, 


Phospho-PAK1 (Ser144/Ser199/Ser204/Thr423)/PAK2 

(Ser20/Ser141/Ser192/Ser197/Thr402) 

Phospho-PAK4 (Ser474)/PAK5 (Ser602)/PAK6 (Ser560) 

guinea pig 

neutrophils 

guinea pig 

neutrophils 

fMLP (1 µM for 30 sec) 

fMLP (1 µM for 30 sec) 

Phospho-Rac1/cdc42 (Ser71) A-431 Serum-starve overnight, 


Phospho-RanBP3 (Ser58) HeLa Serum-starve overnight, 


Phospho-Stathmin (Ser16/Ser38) HeLa Nocadazole (100 ng/ml for 16 hr) 

Phospho-TCTP (Ser46) HT-29 2 mM Thymidine for 16 hr, then 

wash plate, add fresh media and 

Nocodazole (100 ng/ml overnight) 

Phospho-Troponin I (Cardiac) (Ser23/24) 

Primary cardiac Isoproterenol (1 µM for 5 min) 

myocytes 

Phospho-VASP (Ser157/Ser239) A-431 Serum-starve overnight, 

Forskolin (10 µM for 30 min) 

Phospho-Vimentin (Ser56) HeLa Paclitaxel (100 ng/ml for 20 hr) 

Phospho-Vimentin (Ser82) C2C12, C6 Untreated 

Developmental Biology 

Cleaved Notch1 (Val1744) 

MOLT-4, Jurkat Untreated 

Phospho-LRP6 (Ser1490) HeLa Wnt3a-conditioned media for 5 hr 

(Conditioned media from L cells 

transfected with Wnt3a ligand) 

Phospho-NDRG1 (Ser330) Jurkat Calyculin A (100 nM for 20 min) 

Phospho-NDRG1 (Thr346) C2C12 Insulin (30 min) 

Phospho-Numb (Ser276) Ramos TPA (200 nM for 30 min) 

Phospho-Smad1(Ser206/Ser463/Ser465) HeLa Serum-starve overnight, 

BMP (50 ng/ml for 30 min) 

Phospho-Smad2 (Ser245/Ser250/Ser255/Ser465/Ser467) HeLa Serum-starve overnight, 


Phospho-Smad3 (Ser423/Ser425) HT-1080 Serum-starve overnight, 

TGF-β (10 ng/ml for 30 min) 

Phospho-Smad5 (Ser463/Ser465) HeLa Serum-starve overnight, 


#12052 

#12052 



241



Control 

Treatments 

by Target 






Phospho-Smad8 (Ser426/Ser428) HeLa Serum-starve overnight, 


Phospho-YAP (Ser127) HeLa Untreated 

Phospho-β-Catenin (Ser33/Ser37/Thr41/Ser45) SW480 Untreated 

Phospho-β-Catenin (Ser552/Ser675) SK-N-MC Forskolin (50 µM for 30 min) 

Immunology and Inflammation 

Cleaved IL-1β (Asp116) THP-1 Differentiate with TPA (200 nM 

for 24 hr), rest for 24 hr, then 

treat with LPS (1 µg/ml for 24 hr). 

Protein is secreted into media. 

Phospho-5-Lipoxygenase (Ser663) 

mouse & Untreated 

rat brain 

Phospho-AML1 (Ser249) Jurkat, HEL Untreated 

Phospho-BLNK (Tyr96) Ramos Anti-IgM (12 µg/ml for 2 min) 

Phospho-Btk (Ser180) THP-1 Serum-starve overnight, 

H 2 O 2 (2 mM for 2 min) 

Phospho-Btk (Tyr223) Ramos Anti-IgM (12 μg/ml for 10 min) 

Phospho-CD19 (Tyr531) Ramos Anti-IgM (12 μg/ml for 2 min) 

Phospho-CrkL (Tyr207) K-562 Untreated 

Phospho-HS1 (Tyr397) Ramos Serum-starve overnight, 

IgM (12 µg/ml for 10 min) 

Phospho-IKKα/β (Ser176/Ser180) HeLa hTNF-α (20 ng/ml for 10 min) #9243 

Phospho-IKKγ (Ser376) HeLa hTNF-α (20 ng/ml for 10 min) #9243 

Phospho-IRAK1 (Thr209/Ser376/Thr387) HeLa, 293, Untreated 

MCF7 

Phospho-IRF-3 (Ser396) Raw 264.7 LPS (1 μg/ml for 2 hr) 

Phospho-IκBα (Ser32/36) HeLa hTNF-α (20 ng/ml for 5 min) #9243 

Phospho-IκBβ (Ser19/Ser23) HeLa hTNF-α (20 ng/ml) plus 


Phospho-IκBε (Ser18/Ser22) HeLa hTNF-α (20 ng/ml) plus 


Phospho-Jak1 (Tyr1022/1023) HT-29 Serum-starve overnight, 

IL-4 (100 ng/ml for 5–10 min) 

Phospho-Jak2 (Tyr1007/Tyr1008) TF-1 Serum-starve overnight, GM-CSF 

(25 ng/ml for 15 min); GM-CSF 

(2 ng/ml) should be included during 

growth and serum-starvation 

Phospho-Jak2 (Tyr221/Tyr1007/Try1008) CTLL-2 Untreated 

Phospho-LAT (Tyr171/Tyr191) Jurkat Anti-CD3 (10 μg/ml for 2 min) 

Phospho-Lck (Tyr505) Jurkat Anti-CD3 (10 μg/ml for 2 min) 

#9243 

#9243 

Phospho-Lyn (Tyr507) Ramos Serum-starve overnight, 

anti-IgM (12 μg/ml for 2 min) 

Phospho-NF-κB p105 (Ser933) HeLa hTNF-α (20 ng/ml for 5 min) #9243 

Phospho-NF-κB p65 (Ser276) Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-NF-κB p65 (Ser468/Ser536) HeLa hTNF-α (20 ng/ml for 5 min) 

Phospho-p40phox (Thr154) THP-1 Serum-starve overnight, 


Phospho-RelB (Ser552) Jurkat TPA (200 nM for 30 min) 

Phospho-RIP2 (Ser176) THP-1 Treat overnight with TPA (50 ng/ml), 

then culture in fresh medium 48 hr 

to form differentiated macrophages. 

Induce with LPS (1 µg/ml for 10 min). 

Phospho-SLP76 (Ser376) Jurkat Anti-CD3 (10 μg/ml for 2 min) 

Phospho-SHIP1 (Tyr1020) Ramos Anti-human IgM (12 µg/ml for 2 min) 

Phospho-SHIP2 (Tyr986/Tyr987/Tyr1135) K-562 Untreated 

Phospho-Stat1 (Tyr701/Ser727) HeLa Serum-starve overnight, 

IFN-α (100 ng/ml for 5 min) 

#9173 


Phospho-Stat2 (Tyr690) HeLa Serum-starve overnight, 


Phospho-Stat3 (Tyr705/Ser727) HeLa Serum-starve overnight, 

#9133 


Phospho-Stat4 (Tyr693) NK-92 Cytokine-starve overnight, IL-2 

(10 ng/ml for 15 min); IL-2 (5 ng/ml) 

should be included during growth 


#9353 



IL-4 (100 ng/ml for 15 min) 

Phospho-Tyk2 (Tyr1054/Tyr1055) HeLa Serum-starve overnight, 


Phospho-Zap-70 (Tyr319/Tyr493)/Syk (Tyr352) Jurkat Anti-CD3 (10 ng/ml for 2 min) 

MAP Kinase Signaling 

Phospho-A-Raf (Ser299) HeLa Serum-starve overnight, 


#9160 

Phospho-ATF-2 (Thr69/Thr71) NIH/3T3 Anisomycin (25 μg/ml for 30 min) #9223 

Phospho-B-Raf (Ser445) HeLa Serum-starve overnight, 

#9160 


Phospho-Bcr (Tyr177) K-562 Untreated (K-562 cell line 

contains Bcr-Abl fusion) 

Phospho-c-Abl (Tyr89/Tyr204/Tyr245/Tyr412/Thr735) K-562 Untreated (K-562 cell line 

contains Bcr-Abl fusion) 

Phospho-c-Fos (Ser32) HeLa Serum-starve overnight, 

TPA (200 nM for 4 hr) 

Phospho-c-Jun (Ser63/Ser73/Thr91/Ser243) NIH/3T3 UV (40 mJ/cm 2 ), 30 min recovery #9263 

Phospho-c-Jun (Thr93) NIH/3T3 Untreated 

Phospho-c-Raf (Ser259/Ser289/Ser296/Ser301/Ser338) HeLa Serum-starve overnight, 


Phospho-DUSP1/MKP1 (Ser359) HeLa ALLN (50 µM for 5–6 hr) 

Phospho-Elk-1 (Ser383) 

recombinant 

protein 

GST-Elk1 fusion protein 

phosphorylated in vitro by Erk1/2 

Phospho-Erk5 (Thr218/Tyr220) NIH/3T3 Serum-starve overnight, PDGF 

(100 ng/ml for 5 min); IP with 

#12950 prior to western 

Phospho-FRA1 (Ser265) HeLa Serum-starve overnight, 

TPA (200 nM for 4 hr) 

Phospho-FRS2-α (Tyr196/Tyr436) NIH/3T3 Serum-starve overnight, hFGF 

basic/FGF2 (100 ng/ml for 10 min) 

Phospho-GRB10 (Tyr67) H-4-II-E Serum-starve overnight, 

HGF (40 ng/ml for 10 min) 

Phospho-KSR1 (Ser392) HeLa Untreated 

#9160 

#9183 

Phospho-MAPKAPK-2 (Thr222/Thr334) HeLa Serum-starve overnight, UV 

(40 mJ/cm 2 ), 20 min recovery 

Phospho-MEK1 (Thr286/Ser298) HeLa Serum-starve overnight, 

#9160 

Nocodazole (0.2 μg/ml for 16 hr) for 

Thr286; TPA (200 nM for 15 min) 

for Ser298 

Phospho-MEK1/2 (Ser217/Ser221) HeLa Serum-starve overnight, TPA #9160 


Phospho-MKK3/MKK6 (Ser189/Ser207) NIH/3T3 UV (40 mJ/cm 2 ), 30 min recovery #9233 

Phospho-MKK7 (Ser271/Thr275) U-937 Sorbitol (400 mM for 15 min) 

Phospho-MSK1 (Ser376/Thr581) 293 UV (40 mJ/cm 2 ), 30 min recovery 

Phospho-p38 MAPK (Thr180/Tyr182) C6 Anisomycin (25 μg/ml for 20 min) #9213 

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Jurkat TPA (200 nM for 10 min) #9194 

Phospho-p90RSK (Thr359/Ser363/Ser380/Thr573) HeLa TPA (200 nM for 30 min) #9160 

Phospho-PTPα (Tyr789) 

293, COS-7, Untreated 

C6, C2C12 

Phospho-Pyk2 (Tyr402) Jurkat Serum-starve overnight, anti-CD3 

plus anti-CD28 (both 1 µg/ml for 

10 min) 



243



Control 

Treatments 

by Target 






Phospho-RSK2 (Ser227) HeLa TPA (200 nM for 30 min) #9160 

Phospho-RSK2 (Tyr529) SK-N-MC Serum-starve overnight, 

FGF (100 ng/ml for 10 min) 

Phospho-RSK3 (Thr356/Ser360) HeLa Serum-starve overnight, 


Phospho-SAPK/JNK (Thr183/Tyr185) 293 UV (40 mJ/cm 2 ), 

30 min recovery time 

Phospho-SEK1/MKK4 (Ser257/Thr261) HeLa UV (50 mJ/cm 2 ), 

30 min recovery time 

Phospho-SEK1/MKK4 (Ser80) NIH/3T3 PDGF (100 ng/ml for 5 min) 

Phospho-Shc (Tyr239/Tyr240/Tyr317) Hep G2 Serum-starve overnight, 


Phospho-SHP-2 (Tyr542/Tyr580) NIH/3T3 Serum-starve overnight, 


Phospho-Src (Tyr527) COLO201 Untreated 

Phospho-Src Family (Tyr416) COLO201 Serum-starve overnight, 

treat 5 min 20% FBS 

Phospho-SRC-3 (Thr24) MCF7 Untreated 

Phospho-SRF (Ser103) HeLa Serum-starve overnight, 


Phospho-TAK1 (Thr184/Thr187/Ser412) HeLa hTNF-α or IL-1β (20 ng/ml), both 

with Calyculin A (100 nM) for 10 min 

Phospho-TNK1 (Tyr277) KARPAS-299 Untreated (KARPAS-299 cell lines 

contains NPM-ALK fusion) 

Phospho-β-Arrestin 1 (Ser412) 293 Untreated 


Acetyl-CoA Carboxylase C2C12 Untreated 

Phospho-Acetyl-CoA Carboxylase (Ser79) C2C12 Serum-starve overnight, 

AICAR (0.5 mM for 30 min) 

Phospho-AMPKα (Thr172) C2C12 Serum-starve overnight, 


Phospho-AMPKα1 (Ser485) C2C12 Serum-starve overnight, 


Phospho-AMPKβ1 (Ser108/Ser182) C2C12 Serum-starve overnight, 


Phospho-AS160 (Thr642) HeLa Serum-starve overnight, 

hIGF-I (100 ng/ml for 15 min) 

Phospho-ATP-Citrate Lyase (Ser454) NIH/3T3 Serum-starve overnight, 


Phospho-C/EBPα (Ser21) 

differentiated 

3T3-L1 

Phospho-C/EBPα (Thr222/Thr226) U-937 Untreated 

Phospho-C/EBPβ (Ser105) PC-12 Untreated 

Phospho-C/EBPβ (Thr235) 


3T3-L1 

Serum-starve overnight, 

insulin (100 nM for 45 min) 



Phospho-Glycogen Synthase (Ser641) 293 Serum-starve overnight, 


Phospho-HSL (Ser563/Ser565/Ser660) 


3T3-L1 

Isoproterenol (10 µM for 20 min) 

Phospho-IGF-I Receptor β (Tyr1316/Tyr980/Tyr1131/ 

Tyr1135) 

Phospho-IRS-1 (Ser302/Ser307/Ser318/Ser332 

/Ser336/Ser612/Ser636/Ser639/Ser1101) 

293 Serum-starve overnight, 


MCF7 Serum-starve overnight, 


Motif Antibodies 

Acetylated-Lysine COS-7 TSA (0.4 μM for 18 hr) 

Acetylated-Lysine (Ac-K2-100) COS-7 TSA (0.4 μM for 18 hr) 

Cleaved Caspase Substrate HeLa Staurosporine (1 µM for 3 hr) 

Mono-Methyl Arginine HCT116 Untreated 

#9160 

#9253 

#9160 

#9158 

#9158 

#9158 

#9158 

Phospho-(Ser) 14-3-3 Binding Motif Jurkat Calyculin A (100 nM for 30 min) #9273 


Phospho-(Ser) Arg-X-Tyr/Phe-X-pSer Motif Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-(Ser) CDKs Substrate HeLa Nocadazole (1 μg/ml for 12 hr) 

Phospho-(Ser) PKC Substrate HeLa TPA (200 nM for 30 min) #9160 

Phospho-(Ser/Thr) AMPK Substrate 293 Serum starved for 1 hr #9158 

Phospho-(Ser/Thr) ATM/ATR Substrate 293 UV (50 mJ/cm 2 ), 30 min recovery #9253 

Phospho-(Ser/Thr) PDK1 Docking Motif Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-(Ser/Thr) Phe Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-(Ser/Thr) PKD Substrate HeLa TPA (200 nM for 30 min) #9160 

Phospho-(Thr) MAPK/CDK Substrate Jurkat Nocadazole (1 μg/ml for 12 hr) 

Phospho-Akt Substrate (RXRXXS*/T*) Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-PKA Substrate (RRXS/T) Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-Threonine NIH/3T3 PDGF-BB (100 ng/ml for 5 min) 

Phospho-Threonine-X-Arginine Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-Tyrosine Jurkat Pervanadate (1 mM for 30 min) 

Neuroscience 

Phospho-AMPA Receptor (GluR 2) (Tyr869/873/876) rat brain ischemia and reperfusion 

Phospho-APP (Thr668) HeLa Nocodazole (1 µg/ml for 18 hr) 

Phospho-CaMKII (Thr286) HeLa TPA (200 nM for 15 min) #9160 

Phospho-CREB (Ser133) SK-N-MC Forskolin (30 µM) and IBMX 

(0.5 mM for 30 min) 

Phospho-CRMP-2 (Thr514) rat brain Untreated 

Phospho-DARPP-32 (Thr34/Thr75) 

rat & mouse PKA (12,500 U/100 μl for 1 hr) 

brain 

Phospho-Doublecortin (Ser297) fetal rat brain Untreated 

Phospho-Doublecortin (Ser334) 

rat & mouse Untreated 

brain 

Phospho-MAP2 (Ser136/Thr1620/Thr1623) PC-12 Positive control: Nocodazole 

(1 µg/ml for 16 hr) 

Phospho-MAPK/CDK Substrates (PXSP or SPXR/K) HeLa Nocadazole (1 μg/ml for 12 hr) 

Phospho-Merlin (Ser518) 

rat, mouse, & Untreated 

human brain 

Phospho-NMDAR2A (Tyr1246) rat brain Untreated 

Phospho-NMDAR2B (Tyr1070/Tyr1472) rat brain Untreated 

Phospho-PSD93 (Tyr340) rat brain Untreated 

Phospho-PSD95 (Tyr236/Tyr240) rat brain Untreated 

Phospho-Semaphorin 4B (Ser825) MKN-45 Untreated 

Phospho-Synapsin (Ser9) PC-12 NGF (100 ng/ml for 2 hr) 

Phospho-Tau (Ser396) 

mouse & rat Untreated 

brains 

Phospho-TrkA (Tyr490/Tyr674/Tyr785) PC-12 NGF (100 ng/ml for 2–5 min) 

Phospho-TrkB (Tyr516/Tyr706/Tyr707/Tyr816) PC-12 NGF (100 ng/ml for 2–5 min) 

Phospho-Tyrosine Hydroxylase (Ser31/Ser40) 

rat, mouse, & 

human brain, 

SH-SY5Y, PC- 

12, Neuro-2a 

Untreated 

Nuclear Receptor Signaling 

Phospho-Estrogen Receptor α (Ser104/Ser106/Ser118) MCF7 Serum-starve overnight, hEGF 

(100 ng/ml) plus estradiol (100 nM) 

for 30 min 

Phospho-Glucocorticoid Receptor (Ser211) A549 Serum-starve overnight, 

dexamethasone (100 nM for 1 hr) 

Phospho-Nur77 (Ser351) Jurkat TPA (40 nM), A23187 (2 µM), 

both for 4 hr 

Phospho-Progesterone Receptor (Ser190) T-47D Serum-starve overnight, 

promegestone (100 nM for 1 hr) 

#9193 



245





Phospho-Akt (Ser473) Jurkat Untreated or Calyculin A (100 nM 

for 30 min); alternative: serumstarve, 


works for most cell lines 

#9273 

Phospho-Akt (Thr308/Thr450) Jurkat Calyculin A (100 nM for 30 min) #9273 

Phospho-Drosophila p70 S6 Kinase (Thr398) S2 hEGF (100 ng/ml for 30 min) 

Phospho-eNOS (Ser113/Thr495) BAEC Untreated 

Phospho-eNOS (Ser1177) BAEC VEGF (50 ng/ml for 5 min) 

Phospho-FoxO1 (Thr24/Ser256/Ser319) HeLa Serum-starve overnight, 

20% serum for 30 min 

Phospho-FoxO3a (Thr32/Ser318/Ser321/Ser253) HeLa Serum-starve overnight, 


Phospho-FoxO4 (Ser193) HeLa Serum-starve overnight, 


Phospho-Gab1 (Tyr307/Tyr627) Hep G2 Serum-starve overnight, 


Phospho-Gab2 (Ser159/Tyr452) H-4-II-E Serum-starve overnight, 


Phospho-GSK-3α (Ser21) NIH/3T3 Serum-starve overnight, 


Phospho-GSK-3β (Ser9) NIH/3T3 Serum-starve overnight, 


Phospho-GSK-3β (Thr390) HeLa Paclitaxel (100 nM/ml for 20 hr) 

Phospho-mTOR (Ser2448/Ser2481) MCF7 Serum-starve overnight, 

insulin (100 nM for 5–10 min) 

Phospho-p70 S6 Kinase 

(Thr389/Ser371/Thr421/Ser424) 

MCF7 

Phospho-PDK1 (Ser241) Jurkat, HeLa Untreated 

Phospho-PI3K p85 (Tyr458)/p55 (Tyr199) NIH/3T3/Src Untreated 



Phospho-PRAS40 (Thr246) NIH/3T3 Serum-starve overnight, 


Phospho-PTEN (Ser380/Thr382/Thr383) HeLa Untreated 

Phospho-Raptor (Ser792) 293 Oligomycin (0.5 µM for 30 min) 

Phospho-Rictor (Thr1135) HeLa Serum-starve overnight, 


Phospho-SGK1 (Ser78) SK-MEL-28 H 2 O 2 (4 mM for 15 min) 

Phospho-SGK3 (Thr320) MCF7 H 2 O 2 (4 mM for 15 min) 

Phospho-Tuberin/TSC2 

NIH/3T3 PDGF (50 ng/ml for 30 min) 

(Ser939/Ser1254/Thr1462/Tyr1571) 

Phospho-WNK1 (Thr60) HT-29 Serum-starve overnight, 


Phospho-YB1 (Ser102) MCF7 Serum-starve overnight, 


Vesicle Trafficking and Protein Folding 

Phospho-HSP27 (Ser15/Ser78/Ser82) HeLa Serum-starve overnight, UV 


Phospho-HSP90α (Thr5/Thr7) HeLa Serum-starve overnight, UV 


Phospho-PERK (Thr980) AR42J Thapsigargin (1 μM for 20 min) 

#9203 

#9203 


Phospho-Etk (Tyr40) Jurkat Serum-starve overnight, anti-CD3 

(1 μg/ml) plus anti-CD28 (0.5 μg/ml) 

for 10 min 

Phospho-FLT3 (Tyr589/Tyr591/Tyr842/Tyr969) SEM Untreated 

Phospho-HER2/ErbB2 (Tyr877/Tyr1221/Tyr1222/ 

Tyr1248) 

MCF7 


neuregulin (100 ng/ml for 5 min) 

Phospho-HER3/ErbB3 (Tyr1197/Tyr1222/Tyr1289) MCF7 Serum-starve overnight, 


Phospho-HER4/ErbB4 (Tyr984/Tyr1284) MCF7 Serum-starve overnight, 


Phospho-M-CSF Receptor (Tyr699/Tyr723/Tyr809/Tyr923) NKM-1 Untreated 

Phospho-Met (Tyr1003/Tyr1234/Tyr1235/Tyr1349) H-4-II-E Serum-starve overnight, 


Phospho-PDGF Receptor α (Tyr754/Tyr849/Tyr1018) NIH/3T3 Serum-starve overnight, 

PDGF-β (100 ng/ml for 5 min) 

Phospho-PDGF Receptor β 

(Tyr740/Tyr751/Tyr771/Tyr857/Tyr1009/Tyr1021) 

NIH/3T3 

Phospho-Ret (Tyr905) TT Untreated 


PDGF-β (100 ng/ml for 5 min) 

Phospho-Ros (Tyr2274) HCC78 HCC78 cells express a 

SLC3482-Ros fusion protein 

Phospho-VEGF Receptor 2 (Tyr1175/Tyr1212) HUVEC Serum-starve overnight, 

VEGF (100 ng/ml for 5 min) 

Translational Control 

Methyl-PABP1 (Arg455/Arg460) HeLa, C6, 

COS-7 

Untreated 

Phospho-4E-BP1 (Thr37/Thr46/Ser65/Thr70) HeLa Untreated 

Phospho-eEF2 (Thr56) C6 Forskolin (10 µM for 1 hr) 

Phospho-eEF2k (Ser366) 

HeLa, COS-7, Untreated 

NIH/3T3, C6 

Phospho-eIF2α (Ser51) HeLa Thapsigargin (300 nM for 30 min) 

Phospho-eIF4B (Ser422) NIH/3T3 Serum-starve overnight, 

20% calf serum for 20 min 

Phospho-eIF4E (Ser209) MCF7 Serum-starve overnight, 


Phospho-eIF4G (Ser1108) HeLa Serum-starve overnight, 


Phospho-elF4B (Ser406) NIH/3T3 Untreated 

Phospho-Mnk1 (Thr197/Thr202) HeLa Serum-starve overnight, 20% 

serum for 20 min or Anisomycin 

(25 µg/ml for 30 min) 

Phospho-PPIG (Ser374) Jurkat Untreated 

Phospho-S6 Ribosomal Protein 

(Ser235/Ser236/Ser240/Ser244) 

MCF7 

Serum-starve overnight, insulin 

(100 nM for 5–10 min) 

Ubiquitin and Ubiquitin-like proteins 

Phospho-BAP1 (Ser592) HeLa Serum-starve overnight, 

UV (50 mJ/cm 2 ), 1 hr recovery 

Phospho-c-Cbl (Tyr731/Tyr774) K-562 Untreated 

#2904 

#9203 

#9213 

#9203 

Control 

Treatments 

by Target 





Tyrosine Kinase 

Phospho-ALK (Tyr1078/Tyr1096/Tyr1278 

/Try1282/Tyr1283/Tyr1586/Tyr1604) 

KARPAS-299 

Untreated (KARPAS-299 cell 

lines contains NPM-ALK fusion) 

Phospho-Axl (Tyr702) NCI-H1299 Gas6 (400 ng/mL for 5 min) 

Phospho-c-Kit (Tyr703/Tyr719) NCI-H526 Serum-starve overnight, 

hSCF (100 ng/ml for 5 min) 

Phospho-EGF Receptor (Thr669/Tyr845/Tyr998 

/Tyr992/Tyr1045/Ser1046 /Ser1047/Tyr1068 

/Tyr1086/Tyr1148/Tyr1173) 

HT-29 



#5634 



247



Amyloid Plaque and Neurofibrillary Tangle Formation in Alzheimer’s Disease 

Dopamine Signaling in Parkinson’s Disease 

PEN-2 

Aph 

Nicastrin 

Presenilin 

APP 

Intracellular 

Domain 

γ- 

Secretase 

Normal State 

Nuclear translocation 

Transcriptional regulation 

AICD 

Normal 

Axon 

Pruning 

α- 

Secretase 

APP 

Alzheimer’s Disease State 

Apoptosis 

Apoptosis 

Casp-6 

Neurofibrillary 

Tangles 

Tau 

Akt 

Aberrant 

Neuronal 

Death 

p35 

β- 

APP 

Secretase 

Calpain 

CDK5 

p25 

GSK-3α/β 

Destabilized 

Microtubules 

Me 

Hyperphosphorylation 

of Tau 

AICD 

γ- 

APP Secretase 

Exosome-mediated 

Secretion of Tau 

Activation of PKC, 

PKA, Erk2 

ROS 

Formation 

Normal State 

Tyrosine 

Tyrosine 

Hydroxylase 

Dopa 

Decarboxylase 

Dopamine 

L-DOPA 

Parkinson’s Disease State 

JNK 

Apoptosis 

MKK4/7 

mTORC2 

Akt 

Dopamine 

Dopamine 

PINK1 

Cell 

Metabolism 

Parkin 

Survival 

Apoptosis ROS 

LRRK2 Mutation 

DJ-1 

PINK1 Mutation 

α-synuclein 

Aggregation 

Lewy Bodies 

REDD1 

Misfolding 

α-synuclein Ub 

Proteosomal Degradation 

Cell Stress, 

Cytokines 

Environmental Toxins, 

Neurotoxins 

Genetic 

Mutations 

Microglia 

Activation 

Increased 

Neuronal Survival, 

Neutrite Outgrowth, 

Synaptic sAPPα 

Plasticity, 

Cell Adhesion 

sAPPα 

Glucose NMDAR 

Transporters 

Glc 

Na + 

DR6 

N-APP 

AMPAR Acetylcholine 

Receptors: 

nAChR 

mAChR 

Ca 2+ 

Glucose NMDAR AMPAR 

Transporters 

Casp-3,-9 

Apoptosis 

sAPPβ 

p53, Bad, 

Bax production 

and activation 

Oxidative Stress 

Aβ40/42 

nAChR 

mAChR 

Amyloid Plaques 

Aβ Misfolding, 

Aggregation 

Lipid 

Peroxidation 

ROS 

Formation 

Membrane Damage 

Microglia 

Activation 

Inflammatory 

Cytokines 

β,γ 

D2-type 

Gα i 

CDK5 

cAMP 

PP1 

AC 

DARPP-32 

DARPP-32 

Gα s 

D1-type D2-type D1-type 

PKA 

β,γ 

Transcription 

AMPAR 

NMDAR 

Ca 2+ 

Na + 

CREB CREM ATF 

AMPAR 

NMDAR 

Post-Synaptic Signaling Blocked 


Alzheimer’s disease is one of the most common neurodegenerative diseases worldwide. Clinically, it is characterized by the presence of extracellular amyloid plaques and 

intracellular neurofibrillary tangles, resulting in neuronal dysfunction and cell death. Central to this disease is the differential processing of the integral membrane protein APP 

(Amyloid β Precursor Protein) in the normal versus disease state. In the normal state, APP is initially cleaved by α-secretase to generate sAPPα and a C83 carboxy-terminal 

fragment. The presence of sAPPα is associated with normal synaptic signaling and results in synaptic plasticity, learning and memory, emotional behaviors, and neuronal 

survival. In the disease state, APP is cleaved sequentially by β-secretase and γ-secretase to release an extracellular fragment called Aβ40/42. This neurotoxic fragment 

frequently aggregates and results in Aβ40/42 oligomerization and plaque formation. Aβ40/42 aggregation results in blocked ion channels, disruption of calcium homeostasis, 

mitochondrial oxidative stress, impaired energy metabolism and abnormal glucose regulation, and ultimately neuronal cell death. Alzheimer’s disease is also characterized 

by the presence of neurofibrillary tangles. These tangles are the result of hyperphosphorylation of the microtubule-associated protein Tau. GSK-3β and CDK5 are the kinases 

primarily responsible for phosphorylation of Tau, although other kinases such as PKC, PKA, and Erk2 are also involved. Hyperphosphorylation of Tau results in the dissociation 

of Tau from the microtubule, leading to microtubule destabilization and oligomerization of the Tau protein within the cell. Neurofibrillary tangles form as a result of Tau 

oligomerization and lead to apoptosis of the neuron. 


Bossy-Wetzel, E., Schwarzenbacher, R., and Lipton, S.A. (2004) Nat. Med. 10, 2–9. • Chen, J.X. and Yan, S.S. (2010) J. Alzheimers Dis. 2, 569–578. • Claeysen, S., 

Cochet, M., Donneger, R., Dumuis, A., Bockaert, J., and Giannoni, P. (2012) Cell. Signal. 24, 1831–1840. • Marcus, J.N. and Schachter, J. (2011) J. Neurogenet. 25, 

127–133. • Müller, W.E., Eckert, A., Kurz, C., Eckert, G.P., and Leuner, K. (2010) Mol. Neurobiol. 41, 159–171. • Nizzari, M., Thellung, S., Corsaro, A., Villa, V., Pagano, 

A., Porcile, C., Russo, C., and Florio, T. (2012) J. Toxicol. 2012, 187297. • Thinakaran, G. and Koo, E.H. (2008) J. Biol. Chem. 283, 29615–29619. 

Parkinson’s disease is the second most prevalent neurodegenerative disorder. Clinically, this disease is characterized by bradykinesia, resting tremors, and rigidity due to 

loss of dopaminergic neurons within the substania nigra section of the ventral midbrain. In the normal state, release of the neurotransmitter dopamine in the presynaptic 

neuron results in signaling in the postsynaptic neuron through D1- and D2-type dopamine receptors. D1 receptors signal through G proteins to activate adenylate cyclase, 

causing cAMP formation and activation of PKA. D2-type receptors block this signaling by inhibiting adenylate cyclase. Parkinson’s disease can occur through both genetic 

mutation (familial) and exposure to environmental and neurotoxins (sporadic). Recessively inherited loss-of-function mutations in parkin, DJ-1, and PINK1 cause mitochondrial 

dysfunction and accumulation of reactive oxidative species (ROS), whereas dominantly inherited missense mutations in α-synuclein and LRRK2 may affect protein degradation 

pathways, leading to protein aggregation and accumulation of Lewy bodies. Mitochondrial dysfunction and protein aggregation in dopaminergic neurons may be responsible 

for their premature degeneration. Another common feature of the mutations in α-synuclein, Parkin, DJ-1, PINK1, and LRRK2 is the impairment in dopamine release and 

dopaminergic neurotransmission, which may be an early pathogenic precursor prior to death of dopaminergic neurons. Exposure to environmental and neurotoxins can also 

cause mitochondrial functional impairment and release of ROS, leading to a number of cellular responses including apoptosis and disruption of protein degradation pathways. 

There is also an inflammatory component to this disease, resulting from activation of microglia that cause the release of inflammatory cytokines and cell stress. This microglia 

activation causes apoptosis via the JNK pathway and by blocking the Akt signaling pathway via REDD1. 


Dauer, W. and Przedborski, S. (2003) Neuron 39, 889–909. • Girault, J.A. and Greengard, P. (2004) Arch. Neurol. 61, 641–644. • Patten, D.A., Germain, M., Kelly, M.A., 

and Slack, R.S. (2010) J. Alzheimers Dis. 20, 357–367. • Imai, Y. and Lu, B. (2011) Curr. Opin. Neurobiol. 21, 935–941. • Springer, W. and Kahle, P.J. (2011) Autophagy 

7, 266–278. 

© 2009–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Christopher Phiel, Univ. of Colorado, Denver, CO and Prof. Jeff Kuret, Ohio State Univ., Columbus, OH for reviewing this diagram. 


© 2009–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. Jie Shen, Harvard Medical School, Boston, MA, for reviewing this diagram. 


III 

Workflow 

Tools 

The biomedical research workflow is often a multi-dimensional rather 

than linear process. Depending on the question you are trying to answer, 

the process may include surveying a new cell type or disease model to 

identify biologically relevant alterations or performing detailed functional 

analysis of a single protein or post-translationally modified site. 

One of the keys to success is finding the right tool to answer each question 

that emerges in your investigation. In the following pages, we invite 

you to explore several research tools that can deliver valuable information 

and insights as you navigate the various phases of your research projects. 


Explore research tools 

How these tools can benefit your research 

Example data 

Modulation 

pg 266 

Localization & 

Classification 

pg 269 

Screening & 

Quantification 

pg 272 

Profiling 

pg 264 

Monitoring 

pg 274 

Identification 

pg 262 

Research 

Verification 

pg 277 

Discovery 

pg 258 

Customization 

pg 278 

Investigation 

pg 254 

Exploration 

pg 250 


Multiple levels shown of key pathways and structures involved in ER and Golgi-mediated 

trafficking and protein processing, including post-translational modifications. 


249

17 

Section III: Workflow Tools 

Exploration 

using PhosphoSitePlus® online database 

How can I explore what is known about the post-translational 

modifications (PTMs) occurring on my protein of interest 

The PhosphoSitePlus ® (PSP) resource is a comprehensive protein modification knowledgebase, 

including experimentally determined PTMs from peer-reviewed journals and analytical tools that enable 

researchers to explore protein sequences, structures, mutations, pathways, and regulation. Created 

with grant support from the NIH and Cell Signaling Technology (CST), and curated by CST scientists, 

this database is continuously updated and features a dynamic, open, and highly interactive interface. 

It is designed to facilitate the study of the roles of PTMs in normal and pathological cellular processes, 

and to help accelerate the discovery of critical disease biomarkers and drug targets. 

Home Page 

Starting point for interacting with PhosphoSitePlus® 

Users can choose from two types of Simple Searches, three Advanced Searches, and three 

Browsing Interfaces. 

A. Simple Searches for Protein or Kinase Substrates 

In addition to the protein search, which will lead to a Protein Page, the substrate search asks the user 

to enter the name of a kinase and returns a list of experimentally verified in vivo and in vitro sites 

phosphorylated by the selected kinase. The preferred substrate sequences can be summarized and 

viewed as a Sequence Logo. 

B. Advanced Search and Browsing Options 

Search interfaces give users the power to explore what is known about proteins and their PTMs. 

Browse interfaces enable users to retrieve data from thousands of high-throughput (HTP) mass 

spectrometry experiments. 

chapter 17: Exploration 

PhosphoSitePlus ® allows a researcher 

to search for or download: 

• Modified proteins, modification sites and sequences associated with specific protein types, 

molecular weights, protein domains, treatments, tissues, diseases, cell lines, and references 

• Sets of known protein kinase substrates from in vivo and in vitro experiments 

• Specific sequences containing modified residues 

• Scripts to generate molecular structures (PyMOL of DeLano Scientific LLC and Chimera) in which 

all modified residues are labeled and color-coded 

• Datasets of PTMs that overlap known mutation sites (nsSNPs), enabling the researcher to identify 

those that have the potential to rewire signaling pathways 

• Datasets of modification sites observed in MS-MS experiments associated with specific diseases, 

cell lines, or tissues 

• Complete datasets of all modification sites reported to regulate downstream processes 

• Pathway datasets in Cytoscape ® of The Cytoscape Consortium and BioPAX formats 

• Sequence logos and motif analyses of sets of modification sites generated within PSP or 

submitted by the user 

Search Proteins or Sequences by: 

• Name or accession ID 

• Protein type or domain 

• Cellular component 

• Molecular weight range 

• Sequence/motif 

• List of peptides 

Search References by: 

• Author, PubMed ID, or protein name 

Search for Sites by: 

• Response to treatments 

• Tissues, cell line, or cell type 

• Correlation with disease state 

• Protein type, cellular component 

• Protein function or biological process 

Comparative Site Search: 

• Identify sites observed in one 

set of conditions but not others 

• Sequence, motif, or domain 

• Protein molecular weight 

Downloads Datasets: 

• Sites and metadata of major 

modification type 

• Disease mutations and genetic 

variants that overlap PTMs 

• Sites reported to regulate 

downstream processes 

• Sites regulated by specified 

treatments 

• Pathway information in Cytoscape ® 

and BioPAX formats 

Browse and Download HTP 

Data Associated with: 

• Specific diseases 

• Cell lines 

• Tissue 

Sequence Analysis Tools: 

• Sequence Logo Generator 

• Motif Analyzer 

Navigation and Content 

Interactive content is delivered in four increasingly granular interfaces. Navigation between the interfaces 

is seamless. Each page has links back to the Homepage. 

A 

Home Page; is the launch site for 

searching, browsing, static downloads, 

and informational pages. 

Protein Page; opens when the 

user selects the name of a protein 

from a Search Results page 

Home Page 

Protein Page 

Modification Site Page 

B 

Modification Site Page; opens 

when a user clicks on a specific site 

either from the Protein Page or from 

a Search Results page. 

Curated Information Page 

Curated Information Page; 

opens when a user clicks on a 

specific reference on a Modification 

Site Page, and presents the most 

granular information in PSP. 



251


chapter 17: Exploration 

Protein Page 

Provides information about a protein and its PTMs. 

Modification Site Page 

Summarizes information on single site from all records. 

C. Overview: 

• Brief description of protein function 

• Protein type and cellular component 

• Chromosomal location of human 

ortholog 

• Molecular function and 

biological process 

• Accession IDs, synonyms, 

and gene symbols 

• Molecular weight, isoelectric 

point, and pI calculator 

• Associated molecular structures 

and viewer 

• CST pathways and antibodies 

• Structural viewer 

D. Linked Resources Include: 

• Pathways: STRING, Reactome, 

NetworKIN 

• Expression: Protein Atlas, BioGPS 

• Scansite: Predictor of Kinases and 

Interactors 

• KinBase: Kinase Database at The 

Salk Institute 

• Structures: RCSB PDB, Pfam 

• NextGen Protein Resource: neXtProt 

E. Sites Implicated In: 

• Biological and molecular regulation, 

with links to associated site pages 

F. Modification Sites 

and Domains: 

• Zoomable linear diagram 

with sequence 

• Domains and modification 

sites mapped 

• Domain names linked to Pfam 

C 

D 

E 

F 

H 

I 

J 

Curated Information Page 

Experimental details on all sites in a single record. 

H. Site Information: 

•For most sites identified at CST, links 

to MS/MS spectra are included 

• Scansite predictions provide potential 

kinases and binding partners 

• Blast links allow site comparison 

against NCBI, UniProt, or PDB 

I. Experimental Summary 

Sections: 

May include information about how 

the site was experimentally characterized; 

diseases, cell lines and tissues 

in which it was observed; upstream 

control by treatments, receptors and 

other cellular proteins, and enzymes 

that may directly modify the site 

(in vivo and in vitro) 

J. References: 

Information curated from the literature 

contains links to the PubMed entry. 

Information curated from MS/MS 

experiments at CST or other institutions 

indicates the biological sample studied 

and treatments. If performed at CST 

with PTMScan ® Technology, the 

antibody used for peptide enrichment 

prior to MS/MS is indicated. 

Links to the Curated Information Page 

are provided for most modification sites. 

G. Table of Sites, Sequences, 

and References: 

• Modification sites and surrounding 

sequences (+/- 7 AA) from parent 

protein, orthologs, and isoforms 

• Red characters indicate modified 

sites with links to associated records 

• First column (SS): the number 

of records using site-specific, 

low-throughput techniques 

• Second column (MS): the number of 

records using mass spectrometrybased 

high-throughput techniques 

G 

Download PyMOL and/or Chimera script 

Highlight modified residues 

K 

L 

K. Record and Sites: 

Standard bibliographic information 

about the record links to its PubMed 

entry. A list of modification sites 

associated with this record links to 

the the details curated for each site. 

L. Details Curated for 

each Modification Site: 

• Methods used to characterize site 

• Upstream Regulation 

– Kinases, phosphatases, receptors 

and signaling intermediates 

that regulate modification 

– Treatments that regulate 

modification 

• Downstream Regulation 

– Effects on protein function and 

biological processes due to 

modification 

– Protein-protein interactions directly 

influenced by modification 

• Disease Relevance of Modification 



253

18 Investigation 


using motif and post-translational 

modification-specific antibodies 

Motif and post-translational modification (PTM) antibodies 

can help answer two major scientific questions. 

1. How can I measure global changes in PTMs or protein kinase activity in response 

to cellular treatments 

2. How can I investigate PTMs on a protein of interest without complex in vivo labeling 

experiments or where no target-specific antibody is available 

Post-translational modifications of proteins catalyzed by kinases, acetylases, methylases, ubiquitin 

ligases, and other enzymes are key regulators of cell signaling and can be valuable targets for therapeutic 

intervention in disease states. Antibodies recognizing specific protein modification sites (e.g. 

Phospho-Akt, Acetyl-Histone) are well-known and valuable tools for studying PTMs. To augment these 

tools, CST has developed motif and PTM-specific antibodies, a distinct class of PTM reagents that 

have a broader target specificity and can therefore be used for a different set of applications. 

Motif Antibodies 

PTMs catalyzed by a specific modification enzyme are often constrained to a peptide sequence pattern, 

or motif, that reflects the substrate specificity of that enzyme. For example, substrates of the 

protein kinase Akt generally contain an RXRXXS motif in which Akt phosphorylates the serine residue. 

Antibodies that detect the motif RXRXXpS can be used to detect a broad spectrum of Akt substrates. 

Motif antibodies, proprietary to CST, recognize a modified residue in the context of a specific peptide 

sequence motif. These highly specialized antibodies were originally developed for the identification 

and quantification of PTMs catalyzed by kinases from several branches of the human kinome. More 

recently, individual motif antibodies have been developed to recognize several other post-translational 

modifications such as methyl-arginine and caspase cleavage sites. 

PTM-specific Antibodies 

PTM-specific antibodies differ from motif antibodies in that they recognize a specific protein posttranslational 

modification, but are not restricted to a specific sequence motif surrounding the modification 

site. Examples of PTM-specific antibodies from CST include Phospho-Tyrosine (P-Tyr-1000) Rabbit 

mAb #8954 and Acetylated-Lysine (Ac-K 2 -100) Rabbit mAb #9814. Together, motif and PTM-specific 

antibodies provide a robust toolbox for the identification and classification of PTMs and their effects on 

cellular regulation. 

How can motif and PTM-specific antibodies be used 

• Quantitatively identify the PTM alterations of cellular proteins due to up- or down-regulation 

of a specific kinase, acetylase, methylase, nitrosylase, or ubiquitin ligase. 

• Motif and PTM antibodies can be used in early-stage investigations where a high quality 

antibody may not be available for a specific modified protein target. Assay target-specific 

PTM alterations via immunoprecipitation (IP) enrichment of the target protein followed by 

western blot (WB) analysis using the motif or PTM-specific antibody. 

• Screen candidate therapeutics using in vitro kinase and phosphatase assays based on a 

motif or PTM-specific antibody. 

One example of the application of a motif antibody involves the response of Jurkat cells to the PP1 and 

PP2A protein phosphatase inhibitor Calyculin A. The response was studied by 2D gel electrophoresis 

followed by WB analysis using a CST motif antibody recognizing the phospho-serine 14-3-3 binding 

motif. The data reveal a pronounced increase in overall phosphorylation levels. Based on this initial 

study, individual proteins of interest are selected for further study. 

Specific kinases in the human kinome phosphorylate 

target proteins at distinct substrate sequence motifs. 

CHED 

Haspin 

STLK3 

MAST4 

chapter 18: Investigation 

TK 

FGFR2 FGFR3 

TrkC 

EphB2 

TrkB 

FGFR1 

FGFR4 

EphB1 

TrkA 

FLT1/VEGFR1 

ATM 

EphA5 

KDR/VEGFR2 

MuSK ROR2 

Fms/CSFR 

Atypical 

ROR1 

EphB3 

Ret 

Kit 

EphA3 

DDR2 

Mer 

ATM 

EphA4 

DDR1 

Tyro3/ 

Axl 

FLT4 

Sky 

ATR 

PDGFRα 

EphA6 

IGF1R IRR 

FLT3 

PDGFRβ 

mTOR/FRAP 

InsR 

Yes 

EphB4 

Met 

EGFR 

PIKK 

HER2/ErbB2 

Ron 

DNAPK 

Src 

MLK3 

Ros 

SMG1 

EphA7 

ALK 

MLK1 

Lyn 

LTK 

Tie2 

Tie1 

TRRAP 

HCK 

Fyn 

RYK 

HER4 

EphA8 

MLK4 

CCK4/PTK7 

TKL 

MLK2 

Lck 

Fgr 

Ack Tnk1 Tyk2 

Jak1 

HER3 

Jak2 

EphA2 

Jak3 

BLK 

ANKRD3 SgK288 

DLK 

Syk Zap70/SRK 

EphA1 

PYK2/FAK2 

LZK 

FAK 

Lmr1 

ALK4 

ITK 

Lmr2 

C-Raf/Raf1 

FRK 

TGFβR1 

ZAK 

BRaf 

TEC 

EphB6 

KSR 

Srm 

RIPK2 

KSR2 

TXK 

Brk 

BTK 

Lmr3 

ALK7 

IRAK3 

IRAK1 

LIMK1 

ARaf 

BMPR1B 

Etk/BMX 

EphA10 


BMPR1A 

CTK 

RIPK3 

TSK2 

TAK1 

HH498 

ALK1 

CSK CK2a1 

PAK1 

ALK2 

Abl2/Arg 

IRAK2 

ActR2 

Abl Fes 

ActR2B 

STE 

Fer 

RIPK1 

Jak3~b 

TGFβR2 

LRRK2 

MEKK2/MAP3K2 

Jak2~b 

LRRK1 

MISR2 

MEKK3/MAP3K3 

Tyk2~b 

SuRTK106 

IRAK4 

BMPR2 

ASK/MAP3K5 

ANPα/NPR1 

MAP3K8 

Jak1~b 

MAP3K7 

ANPβ/NPR2 

KHS1 

MOS 

KHS2 

HSER 

SgK496 

WNK1 

WNK3 

MEKK6/MAP3K6 

DYRK2 

Mst4 

PBK 

MAP3K4 

DYRK3 

GUCY2D 

WNK2 

DYRK4 

DYRK1A 

NRBP1 

GUCY2F 

NRBP2 MEKK1/MAP3K1 OSR1 

DYRK1B 

WNK4 

MLKL 

PERK/PEK 

SgK307 

SLK 

PKR 

LOK 

HIPK1 HIPK3 

GCN2 SgK424 

TAO1 

SCYL3 TAO2 

HIPK2 

SCYL1 

Tpl2/COT 

SCYL2 

NIK 

TAO3 

PAK1 

CLK4 HIPK4 

PRP4 

HRI 

PAK3 

CLIK1 

PAK2 

IRE1 

CLK2 CLK1 

PAK4 

MAP2K5 

PAK5/PAK7 

CLIK1L 

IRE2 

CLK3 

TBCK 

PAK6 

MAP2K7 

MEK1/MAP2K1 

RNAseL 

GCN2~b 

MEK2/MAP2K2 

TTK 

MSSK1 

SgK071 

KIS 

GRK2 

CMGC 

SRPK2 

MYT1 


SRPK1 

CK2α1 

Wee1 

MAK 

CK2α2 

SgK196 

CDC7 

Wee1B 

CK1δ 

ICK 

PRPK 

TTBK1 

CK1ε 

GSK3β 

MOK 

TTBK2 

GSK3α 

CK1α1 

CDKL3 

CK1α2 

CDKL2 

PINK1 

SgK493 

SgK269 

VRK3 

CK1γ2 

CDKL1 CDKL5 

ERK7 

SgK396 

SgK223 

CDKL4 Erk4 

Slob 

CK1γ1 

Erk3 

SgK110 

PIK3R4 

CK1γ3 

NLK 

SgK069 

Bub1 

Erk5 

SBK 

BubR1 

Erk1/ 

IKKα 

p44MAPK 

IKKβ 

VRK1 

CDK7 

IKKε 

VRK2 

Erk2/ 

Erk2 

CK1 

PLK4 

PITSLRE 

TBK1/NAK 

p42MAPK p38γ 

MPSK1 

JNK1 

p38δ 

JNK2 

TLK2 

JNK3 CDK10 

GAK 

TLK1 

PLK3 

p38β 

AAK1 

CDK8 CDK11 

CAMKK1 

ULK3 

PLK1 

p38α 

PLK2 

CDK4 

CCRK 

BIKE 

CAMKK2 

BARK1/GRK2 

CDK6 

ULK1 


Fused 

GRK5 

PFTAIRE2 

SgK494 

ULK2 ULK4 

GRK6 GRK4 

PFTAIRE1 

CDK9 

Nek6 

RSKL1 

PCTAIRE2 

Nek7 Nek10 

SgK495 

PASK 

PDK1 

RSKL2 

Nek8 

MSK1 

RSK1/p90RSK 

PCTAIRE1 

CDK5 CRK7 

PCTAIRE3 

Nek9 

LKB1 

MSK2 

RSK4 RSK2 

Chk1 

p70S6K 

RSK3 

Nek2 

Akt2/PKBβ 

AurA/Aur2 

p70S6Kβ 

Akt1/PKBα 

cdc2/CDK1 

Nek11 

CDK3 

CDK2 

Nek4 

AurB/Aur1 

Trb3 Pim1 

AurC/Aur3 

Pim2 

LATS1 

Nek3 

Pim1Trb2 

Pim3 

Nek5 

Trad Trio 

LATS2 

NDR1 

Trb1 

Obscn~b 

NDR2 

Nek1 

YANK1 

SPEG~b 

MAST3 

MASTL 

Obscn 

STK33 

YANK2 PKN1 

YANK3 

SPEG 

TTN 

MAST2 

SgK085 

caMLCK 

MAPKAPK2 

skMLCK 

smMLCK 

DRAK2 

DRAK1 

DAPK2 

DAPK3 

DAPK1 

SSTK 

TSSK3 

TSSK1 

TSSK2 

AMPKα2 

AMPKα1 

BRSK2 

CAMK 

BRSK1 

SNARK 

ARK5 

QSK 

SNRK 

NIM1 

SIK 

QIK 

TSSK4 

MELK 

MARK4 

MARK3 

MARK1 

MARK2 

HUNK 

PKD2/PKCµ 

PKD1 

PKD3/PKCν 

MNK1 

MNK2 

RSK4~b 

RSK1~b 

RSK2~b 

RSK3~b 

CASK 

MAPKAPK5 

MAPKAPK2 

MAPKAPK3 

MSK2~b 

MSK1~b 

Chk2/Rad53 

STRAD/STLK5 

STLK6 

CaMKIβ 

CaMKIγ 

MAST1 

DCAMKL3 

DCAMKL1 

DCAMKL2 

VACAMKL 

PhKγ1 

PhKγ2 

PSKH1 

CaMKIIγ 

PSKH2 

CaMKIIα 

CaMKIIβ 

CaMKIIδ 

CaMKIV 

CaMKIα 

CaMKIδ 

GRK7 

HPK1 

GCK 

Akt3/PKBγ 

SGK1 

SGK2 

SGK3 

PKG2 

PKN1/PRK1 

PKG1 

PKN2/PRK2 

PKN3 

PRKY 

PKCδ 

PKCθ 

PRKX 

PKCη 

PKCε 

PKCι 

PKCζ 

PKAγ 

PKAα 

PKCγ 

PKAβ 

AGC 

ROCK1 

PKCα 

ROCK2 

PKCβ 

DMPK 

CRIK 

DMPK2 

MRCKβ 

MRCKα 

Representative phosphorylation motifs from the PhosphoSitePlus ® online resource. Logos for individual motifs are superimposed on the kinase dendrogram developed in collaboration 

between Gerard Manning and Cell Signaling Technology (1). These and over 100 other kinase motifs can be generated for kinases that have 15 or more unique protein substrates reported 

in the literature, and these are curated into the PhosphoSitePlus ® knowledge base (2). 

References 

1. Manning, G., et al. (2002) Science 298, 1912–1934. 

2. Hornbeck, P.V., et al. (2012) Nucleic Acids Res. 40, 261–270. 

NRK/ZC4 

Mst1 

Mst2 

TNIK/ZC2 

MYO3A 

MYO3B 

YSK1 

Mst3 

HGK/ZC1 

MINK/ZC3 

Akt 


www.cellsignal.com/investigation 

255


chapter 18: Investigation 

Treatment with 

Calyculin A induces 

a broad increase in 

phosphorylation at 

phospho-Ser 14-3-3 

substrate motif sites. 

How CST motif and PTM-specific 

antibodies can benefit your research: 

• Multi-pathway Analysis: motif antibodies allow the detection of a broad range of substrates for 

a single kinase, so multiple pathways can be analyzed simultaneously using a single antibody. 

SDS-PAGE 

→ 

• Multiplex Applications: many CST motif antibodies are validated for IP, peptide ELISA (E-P), 

and/or immunohistochemistry (IHC) in addition to WB, enabling flexibility in experimental design 

and the possibility of confirming results via different applications. 

• Proteomic Analysis: CST PTMScan ® services and kits integrate motif and PTM-specific 

antibodies with mass spectrometry (MS) to enable the discovery and analysis of hundreds 

to thousands of PTM sites using standard LC-MS techniques. 

Untreated 

→ 

IPGE 

Calyculin A 

→ 

IPGE 

Phospho-(Ser) 14-3-3 Binding 

Motif Antibody #9601: WB analysis of 

extracts from Jurkat cells, untreated or 

Calyculin A-treated (0.1 µM for 30 min), 

using #9601. Proteins were separated 

by 2-D electrophoresis prior to blotting. 

#9646 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb 

IP † 

RXX(S*/T*) 

(Sepharose ® Bead Conjugate) 

#9611 Phospho-(Ser/Thr) Akt Substrate Antibody W, IP † , IHC-P, E-P (K/R)XX(S*/T*) 

#10001 Phospho-Akt Substrate (RXRXXS*/T*) (23C8D2) Rabbit mAb W, E-P RXRXX(S*/T*) 

#5759 Phospho-(Ser/Thr) AMPK Substrate (P-S/T 2 -102) Rabbit mAb W, IP † , E-P (L/M)XRXX(S*/T*), 

RXX(S*/T*) 

#2909 Phospho-(Ser/Thr) ATM/ATR Substrate (4F7) Rabbit mAb W L(S*/T*)Q 

#2851 Phospho-(Ser/Thr) ATM/ATR Substrate Antibody W, IP † , IHC-P, E-P L(S*/T*)Q 

#6966 Phospho-(Ser/Thr) ATM/ATR Substrate (S*/T*QG) (P-S/T2-100) Rabbit mAb W, IP † (S*/T*)QG, (S*/T*)Q 

#8738 Phospho-(Ser/Thr) CK2 Substrate (P-S/T 3 -100) Rabbit mAb W (S*/T*)DXE 

#9634 Phospho-(Ser/Thr) PDK1 Docking Motif (18A2) Mouse mAb W, IP † , E-P (F/K)XX(F/Y)(S*/T*)(F/Y) 

#9631 Phospho-(Ser/Thr) Phe Antibody W, IP † , E-P (F/Y/W)(S*/T*)(F) 

#9624 Phospho-PKA Substrate (RRXS*/T*) (100G7E) Rabbit mAb W, IP † , E-P (K/R)(K/R)X(S*/T*) 

#9621 Phospho-(Ser/Thr) PKA Substrate Antibody W, IP † , IHC-P, E-P (K/R)(K/R)X(S*/T*) 

#4381 Phospho-(Ser/Thr) PKD Substrate Antibody W, E-P LXRXX(S*/T*) 

Phospho-Tyrosine Application Motif Recognized 

#8954 Phospho-Tyrosine (P-Tyr-1000) Rabbit mAb W, IP † , IF-IC, F Y* 

#9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100) W, IP † , IHC-P, IF-P, Y* 

IF-F, IF-IC, F, E-P 

#9414 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor ® 488 Conjugate) F Y* 

#9415 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Alexa Fluor ® 647 Conjugate) IF-IC, F Y* 

#9417 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Biotinylated) W, IP † , IHC-P Y* 

Antibody recognition 

of the PKA substrate 

motif is phosphorylation-dependent. 

+ + + + 

– – – + – – – – 

Phospho-(Ser/Thr) PKA Substrate 

Antibody #9621: WB analysis of extracts 

from A-431 cells, phosphorylated in vitro 

by protein kinase A (A), Erk2 (B) or cdc2/ 

cyclin A (C), plus or minus PKA inhibitor 

(PKI), using #9621. 

Lanes 

1. PKA 

2. Erk2 

3. cdc2/Cyclin A 

A B C 

Cell Extract 

PKI 

#5465 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (HRP Conjugate) W, E-P Y* 

Motif and PTM-specific antibodies available from CST 

As with our more traditional antibodies, CST’s motif and PTM antibody development team invests significant 

time and resources ensuring that these reagents are highly specific for the motif or PTM of interest. 

These antibodies are validated by a number of methods including WB, peptide arrays, and peptide IP 

followed by LC-MS analysis to confirm that the antibody performs to the highest possible standard. 

#8095 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Magnetic Bead Conjugate) IP † Y* 

#9419 Phospho-Tyrosine Mouse mAb (P-Tyr-100) (Sepharose ® Bead Conjugate) IP † Y* 

#9416 Phospho-Tyrosine Mouse mAb (P-Tyr-102) W, IP † , IHC-P, F, E-P Y* 

Nitro-Tyrosine Application Motif Recognized 

#9691 Nitro-Tyrosine Antibody W Y-NO2 

EGF stimulates 


tyrosine residues in 

many cellular proteins. 

Phospho-Serine Application Motif Recognized 

#9615 Phospho-(Ser) Kinase Substrate Antibody Sampler Kit 

#9606 Phospho-(Ser) 14-3-3 Binding Motif (4E2) Mouse mAb W, IP † , E-P (K/R)XXS*P 

#9601 Phospho-(Ser) 14-3-3 Binding Motif Antibody W, IP † , IHC-P, E-P (K/R)XXS*P 

#9607 Phospho-ATM/ATR Substrate (S*Q) (D23H2/D69H5) Rabbit mAb W, IP † S*Q 

#6967 Phospho-(Ser) PKC Substrate (P-S 3 -101) Rabbit mAb W, IP † , E-P (K/R)XS*X(K/R) 

Acetylated Lysine Application Motif Recognized 

#9814 Acetylated-Lysine (Ac-K 2 -100) Rabbit mAb W, IP † , ChIP, E-P Ac-K 

#6952 Acetylated-Lysine (Ac-K 2 -100) Rabbit mAb (HRP Conjugate) W Ac-K 

#9681 Acetylated-Lysine Mouse mAb (Ac-K-103) W, E-P Ac-K 

#9441 Acetylated-Lysine Antibody W, IP † , IHC-P, IF-IC, Ac-K 

ChIP, E-P 

kDa 

200 

140 

100 

80 

#2325 Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb W, IP † , IHC-P, E-P PXS*P, S*PX(K/R) 

#5501 Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb IP † 

PXS*P, S*PX(R/K) 

(Sepharose ® Bead Conjugate) 

#9477 Phospho-(Ser) CDKs Substrate (P-S 2 -100) Rabbit mAb W, IP † , E-P (K/H)S*P 

#2261 Phospho-(Ser) PKC Substrate Antibody W, IP † , E-P (K/R)XS*(Hyd)(K/R) 

#2981 Phospho-(Ser) Arg-X-Tyr/Phe-X-pSer Motif Antibody W, IP † , E-P RX(F/Y)XS* 

Phospho-Threonine Application Motif Recognized 

#9391 Phospho-Threonine-Proline Mouse mAb (P-Thr-Pro-101) W, IHC-P, E-P T*P 

#5243 Phospho-PLK Binding Motif (ST*P) (D73F6) Rabbit mAb W, IP † , E-P ST*P 

#2351 Phospho-Threonine-X-Arginine Antibody W, IHC-P, E-P T*X(K/R) 

#3004 Phospho-Thr-Pro-Glu (C32G12) Rabbit mAb W, IP † , E-P T*PE, T*P 

#9386 Phospho-Threonine (42H4) Mouse mAb W, IP † , E-P T* 

#9381 Phospho-Threonine Antibody (P-Thr-Polyclonal) W, IP † , E-P T* 

#8781 Phospho-Threonine Antibody (P-Thr-Polyclonal) 

IP † T* 

(Sepharose Bead Conjugate) 

#6949 Phospho-Threonine Antibody (P-Thr-Polyclonal) (HRP Conjugate) W, E-P T* 

Mono-/Di-Methyl-Arginine Application Motif Recognized 

#8015 Mono-Methyl Arginine (Me-R 4 -100) Rabbit mAb W, IP † , E-P Me-R 

#8711 Mono-Methyl Arginine (R*GG) (D5A12) Rabbit mAb W, IP † , E-P Me-RGG 

#13522 Asymmetric Di-Methyl Arginine Motif [adme-R] Rabbit mAb W ADMe-R 

#13222 Symmetric Di-Methyl Arginine Motif [sdme-RG] Rabbit mAb W SDMe-RG 

Ubiquitin Linkage Application Motif Recognized 

#8081 K48-Linkage Specific Polyubiquitin (D9D5) Rabbit mAb W Polyubiquitin formed 

by K48 linkage 

#12805 K48-linkage Specific Polyubiquitin (D9D5) Rabbit mAb (HRP Conjugate) W Polyubiquitin formed 


#4289 K48-linkage Specific Polyubiquitin Antibody W Polyubiquitin formed 


#5621 K63-Linkage Specific Polyubiquitin (D7A11) Rabbit mAb W Polyubiquitin formed 


#12930 K63-linkage Specific Polyubiquitin (D7A11) Rabbit mAb (HRP Conjugate) W Polyubiquitin formed 


60 

50 

40 

30 

– + hEGF 

Phospho-Tyrosine (P-Tyr-1000) Rabbit 

mAb #8954: WB analysis of extracts from 

A-431 cells, untreated or treated with 

Human Epidermal Growth Factor (hEGF) 

#8916 (100 ng/ml, 5 min), using #8954. 

† 

IP denotes suitability for standard IP, not for peptide immunoaffinity enrichment prior to LC-MS/MS. 

Phospho-Serine/Threonine Application Motif Recognized 

#9920 Phospho-(Ser/Thr) Kinase Substrate Antibody Sampler Kit 

#9614 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb W, IP † , IHC-P, E-P RXX(S*/T*) 

#6950 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb (HRP Conjugate) W RXX(S*/T*) 

#8050 Phospho-Akt Substrate (RXXS*/T*) (110B7E) Rabbit mAb 

(Magnetic Bead Conjugate) 

IP † 

RXX(S*/T*) 

PTMScan® Proteomics Services 

Quantitative PTM profiling — see page 258 or visit our website. www.cellsignal.com/discovery 


www.cellsignal.com/investigation 

257

19 


chapter 

Discovery 

using peptide immunoaffinity enrichment and LC-MS/MS 

to study post-translational modifications (PTMs) 

How can I use PTM-specific proteomics to enable discovery 

of novel disease drivers and identification of biomarkers 

How can I profile changes in specific PTM sites on key target 

proteins to explore changes in critical signaling events 

Post-translational modifications such as phosphorylation, acetylation, methylation, ubiquitination, and 

others are critical regulators of protein activity and function. Understanding the role of PTMs in disease 

states and developing novel biomarkers and therapeutics are areas of intense research. However, 

researchers who study PTMs are challenged by their inherent low levels, which make them difficult to 

identify and quantify from whole cell or native samples. 

This challenge can be overcome by using immunoaffinity enrichment to isolate specific peptides 

containing PTMs of interest from a protease-digested cell or tissue extract. LC-MS/MS analysis is then 

employed to identify and quantify the isolated peptides. PTMScan ® Technology, proprietary to CST, utilizes 

the specificity of PTM-specific and motif antibodies to enrich target peptides from the background 

of non-modified endogenous peptides, enabling the identification of modified proteins that otherwise 

may not be detected. Hundreds to thousands of post-translationally modified peptides can be identified 

and quantified in a single experiment. This approach can be used to study modifications such as 

phosphorylation, acetylation, methylation, ubiquitination, succinylation, or caspase-mediated cleavage. 

The highly conserved nature of PTMs and substrate motifs across many different species also makes 

this approach applicable to many different model systems. 

PTM Site Discovery and Profiling 

• Discover candidate biomarkers linked to activation or repression of a specific class of PTMs. 

• Perform a comprehensive proteomic survey of thousands of PTM sites associated with a wide 

variety of organisms or a disease model system, integrating the results into known signaling 

pathways or helping to define novel signaling networks. 

• Detect substrates of novel signaling proteins (kinases, phosphatases, ubiquitin ligases, 

deubiquitinases, acetyl transferases, methyl transferases, or succinyl transferases). 

• Profile and quantify global effects of a candidate therapeutic on a specific type of PTM, 

identifying nodes of interest for further study. 

• Understand how cross-talk among various PTMs (phosphorylation, ubiquitination, acetylation, 

methylation) is related to a particular biological response or involved in cell development or 

differentiation. 

• Analyze downstream effects of targeted gene silencing on signaling and potential activation 

of alternative pathways. 

• Identify protein-protein interaction binding partners along with their corresponding PTMs. 

Discover Low Abundance PTM Sites 

Immunoaffinity enrichment allows you to identify low abundance modifications by selectively capturing 

the PTM in the context of the motif of interest from a population of endogenous peptides. The specificity 

of immunoaffinity enrichment complements that of IMAC, a charge-based metal affinity method 

commonly used for phospho-peptides. IMAC is driven by general coordination chemistry dictated by 

the immobilized metal ion (1). IMAC can therefore lead to enrichment of peptides from more abundant 

proteins for subsequent LC-MS/MS analysis, but not so readily for less abundant phospho-peptides. 

Complementary methods: immunoaffinity 

and IMAC enrichment for phospho-peptides. 

A total of 27,372 unique phosphopeptides 

were isolated from MKN-45 

cells using both IMAC and motif 

antibody enrichment strategies. In 

this study, distinct sets of modified 

peptides were identified by the two 

affinity enrichment methods, with 

only 5.6% overlap between the MS/ 

MS identified peptides. 

Identification of Biomarkers 

PTMScan Technology has been used to identify novel biomarkers in a number of different model systems 

(2–6). One example of the application of PTMScan Technology was conducted in a large-scale 

survey of tyrosine kinase activity in lung cancer, performed at CST. For this study, we used a phosphotyrosine 

motif antibody to analyze changes in phosphorylation across the proteome in non-small cell 

lung carcinoma (NSCLC) cell lines and tissues. 

We surveyed the phospho-tyrosine status of receptor tyrosine kinases (RTK) and non-receptor tyrosine 

kinases in 41 NSCLC cell lines and over 150 NSCLC tumors. Over 50 tyrosine kinases and more than 

2,500 downstream substrates that play roles in NSCLC growth and progression were identified. Two 

very exciting findings from this study were the identification of novel ALK and ROS1 C-terminal fusion 

proteins (EML4-ALK, CD74-ROS, SLC34A2-ROS) in some NSCLC cell lines and tumors (7). Further 

investigation of ALK and ROS1 in other cancers led to the identification of a novel FN1-ALK fusion 

protein in ovarian cancer and a FIG-ROS1 fusion in cholangiocarcinoma (8,9). 

As one consequence of this study, a companion diagnostic immunohistochemistry (IHC) assay for 

NSCLC was developed. In 2011, the diagnostic IHC assay was approved in the U.S. for patients with tumor 

samples that stain positive for ALK fusion protein expression for crizotinib treatment of NSCLC (10). 

ALK fusion protein 

detection in human 

lung carcinoma 

ALK (D5F3 ® ) XP ® Rabbit mAb #3633: 

IHC analysis of paraffin-embedded 

human lung carcinoma with high (left) 

and low levels (right) of ALK fusion 

protein expression using #3633. 

All Antibodies* 

(13,480) 

Carcinoma-associated fusion proteins 

discovered using PTMScan ® Technology 

5.6% 

IMAC 

(13,892) 

*Phospho-Tyrosine, Basophilic Mix, Proline Mix, and Serine/Threonine Mix 

19: Discovery 

Discovery workflow 

for phospho-tyrosine 

modifications 

altered in NSCLC 

Cell or Tissue Samples 

Kinase Family Targeting 

Immunoprecipitation 

Using Motif Antibody 

LC-MS/MS and 

Bioinformatic Analysis 


www.cellsignal.com/discovery 

259


chapter 19: Discovery 

How do I select the most relevant PTM to study 

The first step to discovery of novel PTM alterations in response to disease states or drug treatments 

is selecting the appropriate class of modifications for enrichment. If you are not certain which specific 

classes of modifications are altered in your experimental system, you can first perform a series of 

western blot analyses employing a panel of motif antibodies that detect a range of different PTMs 

(KinomeView ® ). 

What type of data can I expect 

from PTMScan ® kits and services 

The data set generated by a PTMScan service experiment includes not only quantification of PTM 

changes, but also the identity of each protein and specific location of each modification site. For ease 

of interpretation, the experimental results can be viewed at a global level as a scatter plot, as a detailed 

table view (below), or as a domain map for a selected protein. Using the appropriate LC-MS/MS platforms 

and informatics processing, the PTMScan kits can be used to generate comparable results. 

Identify Key Targets from Thousands of Peptides 

The process of data refinement can be illustrated by a simple scenario. A typical PTMScan proteomic 

analysis focused on a single class of PTM might identify 1,000–3,000 unique modified peptides. Of 

these, perhaps 50–100 might exhibit a clear change in modification that correlates with tissue type or 

with the expected biology and pathway associated with the cell treatment employed in the study. With 

further WB and/or ELISA experimentation, you can shorten this list of candidate PTM sites to a limited 

subset of actionable targets (1,4,6). 

Identify 1,000-3,000 unique PTM-containing peptides 

Adjust signal:noise cutoff 

to optimize results 

Representative PTMScan ® data, revealing the protein identity, 

site location, and relative abundance for each PTM detected. 

Normalized Fold Change 

SU11274 vs. 

DMSO Control 

Staurosporine 

vs. DMSO Control 

Protein 

Name Site -7/+ 7 Peptide Upstream Kinase 

-5.0 -4.6 EphA2 897 RVSIRLPsTSGSEGV LPS*T*SGSEGVPFR Akt1 

-13.6 -2.1 FOXO1A 319 TFRPRTSsNASTISG TSS*NASTISGR Akt1 

-158.0 -7.2 FOXO4 32 QSRPRSCtWPLPRPE SCT*WPLPRPEIANQPSEPPEVEPDLGEK Akt1 

-3.4 1.8 QIK 358 DGRQRRPsTIAEQTV RPS*TIAEQTVAK Akt1, Akt2 

-13.3 -29.4 S6 235, 

236, 

240 

-7.0 -24.5 S6 236, 

240 

IAKRRRLsSLRASTS RLS*S*LRAS*TSK Akt1, Akt2, 

P70S6KB, PKACA, 

PKCA, PKCD 

AKRRRLSsLRASTSK RLSS*LRAS*TSK Akt1, Akt2, 

P70S6KB, PKACA, 

PKCA, PKCD 

2.6 1.1 BRAF 365 GQRDRSSsAPNVHIN SSS*APNVHINTIEPVNIDDLIR Akt1, Akt3 

-7.0 -9.4 GSK3B 9 SGRPRTTsFAESCKP TTS*FAESCKPVQQPSAFGSMK Akt1, AurA, 

CAMK2B, GSK3B, 

KHS1, PKACA, 

PKCA 

-5.3 N.D. GSK3B 9, 21 SGRPRTTsFAESCKP TTS*FAESCKPVQQPS*AFGSMK Akt1, AurA, 

CAMK2B, GSK3B, 

KHS1, PKACA, 

PKCA 

-21.3 -3.0 PEA-15 116 KDIIRQPsEEEIIKL DIIRQPS*EEEIIK Akt1, CAMK2A, 

CK2A1 

-2.1 -2.9 GSK3A 21 SGRARTSsFAEPGGG TSS*FAEPGGGGGGGGGGPGGSASGPGGTGGGK Akt1, CAMK2B, 

PKACA, PKCA, 

PKCB 

-10.3 -1.8 RANBP3 126 VKRERTSsLTQFPPS TSS*LTQFPPSQSEER Akt1, ERK1, RSK2, 

p90RSK 

2.7 2.5 eIF4B 422 RERSRTGsESSQTGT TGS*ESSQTGTSTTSSR Akt1, p70S6K, 

p90RSK 

4.8 2.5 eIF4B 422, 

425 

RERSRTGsESSQTGT TGS*ESS*QTGTSTTSSR Akt1, p70S6K, 

p90RSK 

Table view presentation of data from PTMScan ® analysis of MKN-45 cells treated with SU11274 or staurosporine. Shown 

are representative data for the basophilic Akt substrate motif RXRXX(s/t) or RXX(s/t). Relative abundance changes of 2.5-fold or greater 

(treated versus control) for phosphorylated peptides are indicated by green (increase) or light red (reduction) highlighting. Incrementally 

darker highlighting in this image indicates abundance changes in the ranges of 2.5- to 24.9-fold and of greater than 25-fold, respectively. 

Analyze in context of known biology 

of treatment or system 

Map results using protein 

interaction database service 

to reveal novel pathways 

WB and/or 

ELISA validation 

Potential 

key nodes 

(

20 


chapter 

Identification 

using Chromatin IP (ChIP) to identify protein-DNA interactions 

In a second example, the Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb #9145 and SimpleChIP ® 

Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 are used to identify promoters with Stat3 binding 

sites. As shown in the figure, the active form of the Stat3 transcription factor—Phospho-Stat3 

(Tyr705)—binds the c-Fos and IRF-1 promoters, but not the negative control α satellite repeat element. 

20: IDENTIFICATION 

Acetylation of 

histone H3 at Lys27 is 

associated with active 

regions of the genome. 


35 

30 

25 

20 

15 

10 

5 

0 

GAPDH 

RPL30 AFM α Satellite 

SimpleChIP ® Enzymatic Chromatin IP 

Kit (Magnetic Beads) #9003: ChIPs were 

performed with cross-linked chromatin 

from 4 x 10 6 HeLa cells and either 5 μl 

of Acetyl-Histone H3 (Lys27) (D5E4) XP ® 

Rabbit mAb #8173 or 2 μl of Normal Rabbit 

IgG #2729 using #9003. The enriched 

DNA was quantified by real-time PCR using 

SimpleChIP ® Human GAPDH Exon 1 Primers 

#5516, SimpleChIP ® Human RPL30 Exon 3 

Primers #7014, SimpleChIP ® Human AFM 

Intron 1 Primers #5098, and SimpleChIP ® 

Human α Satellite Repeat Primers #4486. 

The amount of immunoprecipitated DNA in 

each sample is presented as a percent of 


Acetyl-Histone H3 (Lys27) 

(D5E4) XP ® Rabbit mAb #8173 


How can I analyze the complex interactions 

between chromatin and the proteins that bind to it 

Chromatin immunoprecipitation (ChIP) is a powerful tool for analyzing protein-DNA interactions within 

the natural chromatin context of the cell. The study of these interactions is critical for understanding 

the transcriptional regulators and epigenetic modifications central to the regulation of gene expression. 

The ChIP method identifies DNA-protein interactions 

using a procedure that can be summarized as follows: 

1. Chemical cross-linking of proteins to DNA in cells or tissues 

2. Tissue disruption and DNA fragmentation to the nucleosome level 

3. Immuno-enrichment of target protein (e.g. histone, transcription factor) 

and associated DNA sequences 

4. DNA purification and analysis 

Quantitative PCR (qPCR) Analysis 

Traditional ChIP, which analyzes the captured DNA by qPCR, is used to test interactions between known 

chromatin-associated proteins and known genetic loci, using an antibody specific to a protein of interest 

and PCR primers to specific genes or promoter regions. ChIP can also be used to study epigenetic 

changes using histone acetylation- or methylation-specific antibodies. 

Sequencing Analysis 

The ChIP method can be combined with sequencing (ChIP-seq) to identify novel regions of the genome 

associated with a particular transcription factor, chromatin regulator, or histone modification. Unlike traditional 

ChIP that requires a predetermined idea of the loci under investigation because primers to that 

region are necessary for subsequent DNA analysis by PCR, in ChIP-seq, all captured DNA fragments can 

be identified and analyzed directly by next generation sequencing. This powerful technique is extremely 

useful for large-scale epigenomic mapping and allows the researcher to take a genome-wide approach 

in the identification of loci associated with a particular transcription factor or histone modification. 

ChIP assays can be used to: 

• Identify multiple proteins associated with a specific region of the genome, 

or many regions of the genome associated with a particular protein 

• Determine the specific order of recruitment of various protein factors to a gene promoter 

• Identify active and inactive regions of the genome through 

associations with specific histone modifications 

• Analyze binding of transcription factors, transcription co-factors, 

DNA replication factors, and DNA repair proteins 

• Study epigenetic aberrations associated with disease 

For example, the SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 from Cell Signaling 

Technology was used to investigate genetic loci associated with acetylated-histone H3 at Lys27. As 

shown in the graph, the ChIP-validated Acetyl-Histone H3 (Lys27) (D5E4) XP ® Rabbit mAb isolates DNA 

fragments containing the active GAPDH and RPL30 genes, but not the inactive AFM gene or α satellite 

repeat element, illustrating that this histone modification associates with active regions of the genome. 

SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003: 

ChIPs were performed with cross-linked chromatin from 4 x 10 6 Hep G2 cells 

starved overnight and treated with IL-6 (100 ng/ml) for 30 min, and either 10 μl 

of Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb #9145 or 2 μl of Normal 

Rabbit IgG #2729 using #9003. The enriched DNA was quantified by real-time 

PCR using human IRF-1 promoter primers, SimpleChIP ® Human c-Fos Promoter 

Primers #4663, and SimpleChIP ® Human α Satellite Repeat Primers #4486. The 

amount of immunoprecipitated DNA in each sample is presented as a percent of 


How SimpleChIP ® and SimpleChIP ® 

Plus Kits can benefit your research: 

• Higher Chromatin Quality: enzymatic digestion of chromatin is milder than sonication, 

better preserving chromatin integrity and improving detection of low-abundance targets 

(transcription factors, co-factors) 


10 

8 

6 

4 

2 

0 

c-FOS 

IRF-1 

α Satellite 

• Appropriate Controls: reliably monitor ChIP efficiency in both active and inactive chromatin 

with included positive control Histone H3 antibody 

• Sample Flexibility: analyze chromatin from cultured cells or tissue samples using 

SimpleChIP ® Plus Kits 

• Rigorously Validated Antibodies: antibody specificity, sensitivity, and ChIP-optimized 

concentrations are key contributors to a successful ChIP experiment 

ChIP-Seq analysis of transcription factor TCF4 binding and 

Tri-Methyl Histone H3 (Lys4) enrichment at several gene loci. 

Number of sequencing reads 

Number of sequencing reads 

50 

25 

0 

80 

40 

0 

50 

25 

0 

80 

40 

0 

A 

C 

CCND1 

AXIN2 

ACTG1 

SimpleChIP ® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005: ChIPs were performed with cross-linked chromatin from 4 x 10 6 

HCT 116 cells and either 10 μl of Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (upper) or 10 μl of TCF4 (C48H11) Rabbit mAb #2569 

(lower), using #9005. DNA sequencing libraries were generated using the NEBNext ® ChIP-Seq Library Prep Master Mix Set for Illumina (E6240) 

and 10 ng of DNA from each IP. The DNA libraries were sequenced using the Illumina MiSeq Sequencer and the obtained sequences were mapped 

to the UCSC Human Genome Assembly (hg19). The data was visualized using Integrative Genomics Viewer (IGV) and this figure shows enrichment 

of H3K4me3 and TCF4 binding at the CCND1 (A), c-Myc (B), AXIN2 (C) and ACTG1 (D) gene loci. 

B 

MYC 

D 

The Phospho-Stat3 

transcription factor 

binds to c-Fos and 

IRF-1 promoters. 


(D3A7) XP ® Rabbit mAb #9145 



www.cellsignal.com/identification 

263

21 


Profiling 

using antibody arrays 

How can you profile the signaling pathways and specific 

protein modifications at work in your experimental system 

An antibody array allows you to simultaneously profile and quantify multiple phosphorylation sites or 

other pathway readouts in a high throughput and economical way. Arrays must contain high quality 

antibody pairs, carefully selected for coverage of the most relevant targets, in order to provide relevant 

and accurate data. 

Antibody arrays can be used to: 

• Characterize a new cell line or disease model 

• Analyze possible effects of a new cellular stimulus 

• Investigate compensatory pathways used by tumors to evade targeted therapies 

• Identify off-target effects of small molecule or biologic therapeutics 

For example, in the simple experiment (seen right), a single variable—in this case, human IGF-I treatment—is 

monitored for effects on signaling nodes within the Akt pathway using the PathScan ® Akt 

Signaling Antibody Array from CST. The data show that treatment with a single dose of human IGF-I 

changes the levels of phosphorylation in Akt (Thr308 and Ser473), S6 Ribosomal Protein (Ser235/236), 

GSK-3α (Ser21), and several other proteins. 

However, experimental designs are not always simple; answering more refined follow-up questions 

may require you to test multiple biological stimulators, dosages, time points, or cell samples. Antibody 

array kits from CST come with 2 slides containing up to 16 pads each, allowing for comprehensive 

experimental design. 

How PathScan ® Antibody Arrays can benefit your research: 

• Multiplex Format: quickly analyze dozens of highly relevant signaling nodes 

across multiple pathways simultaneously 

• Multiple Array Pads: test up to 32 experimental variables in parallel 

• Calibrated Dynamic Range: permits detection of low-abundance targets 

without interference from high-abundance readouts 

• Rigorously Validated Antibody Pairs: generate accurate data and minimize background 

• Low Sample Volume: use as little as 50 μl of sample per pad 

Effect of human IGF-I on multiple downstream Akt signaling pathway nodes 

RFU (x10 4 ) 

75 

60 

45 

30 

15 

0 

1 

1. Akt (Thr308) 

2. Akt (Ser473) 

3. S6 (Ser235/236) 

MCF7 

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 

4. AMPKa (Thr172) 

5. PRAS40 (Thr246) 

6. mTOR (Ser2481) 

Akt Antibody Array target map 

Targets 

Phosphorylation Site 

+ Positive Control N/A 

– Negative Control N/A 

1. Akt Thr308 

2. Akt Ser473 

3. S6 Ribosomal Protein Ser235/236 

4. AMPKa Thr172 

5. PRAS40 Thr246 

6. mTOR Ser2481 

7. GSK-3a Ser21 

8. GSK-3b Ser9 

9. p70 S6 Kinase Thr389 

10. p70 S6 Kinase Thr421/Ser424 

11. Bad Ser112 

12. RSK1 Ser380 

13. PTEN Ser380 

14. PDK1 Ser241 

15. Erk1/2 Thr202/Tyr204 

16. 4E-BP1 Thr37/46 

1 

3 

9 

15 

7. GSK-3a (Ser21) 

8. GSK-3b (Ser9) 

9. p70 S6K (Thr389) 

MCF7 + hIGF-I 

10. p70 S6K 

(Thr421/Ser424) 

11. Bad (Ser112) 

2 

7 

10 

12. RSK1 (Ser380) 

13. PTEN (Ser380) 

14. PDK1 (Ser241) 

+ 1 1 2 2 + 

3 3 4 4 5 5 

6 6 7 7 8 8 

9 9 10 10 11 11 

12 12 13 13 14 14 

+ 15 15 16 16 – 

15. Erk1/2 

(Thr202/Tyr204) 

16. 4E-BP1 

(Thr37/46) 

chapter 21: Profiling 

PathScan ® Akt Signaling 

Antibody Array Kit (Fluorescent 

Readout) #9700: MCF7 cells were 

grown to 85% confluency and then 

serum starved overnight. Cells were 

either untreated or treated with 

Human Insulin-like Growth Factor I 

(hIGF-I) #8917 (100 ng/ml, 20 min). 

Cell extracts were prepared and 

analyzed using #9700. 

MCF7 

MCF7 + hIGF-I 

Images were acquired using the 

LI-COR ® Biosciences Odyssey ® 

imaging system. This graph displays 

pixel intensity quantified using the 

LI-COR ® Image Studio v2.0 array 

analysis software. 

Using PathScan ® Akt 

Signaling Antibody Array Kit 

(Fluorescent Readout) #9700. 

Antibody arrays are 

based upon the sandwich 

immunoassay principle. 

PathScan ® Antibody Array Kits Chemiluminescent Fluorescent 

Akt Signaling #9474 #9700 

EGFR Signaling #12622 #12785 

Immune Cell Signaling #13792 #13788 

Intracellular Signaling #7323 #7744 

RTK Signaling #7982 #7949 

Stress and Apoptosis Signaling #12856 #12923 

Th1/Th2/Th17 Cytokine #13047 #13124 






www.cellsignal.com/profiling 

265

22 


Modulation 

using chemical activators and inhibitors, 

cytokines, and growth factors 

Inhibition of Signaling Using Chemical Modulators 

gefitinib 

Wortmannin 

LY294002 

GF Growth Factors, Insulin, etc. 

membrane 

chapter 22: Modulation 

How can I activate or inhibit specific signaling nodes 

within a cell to study its biology or role in disease 

Chemical modulators, cytokines, and growth factors are important tools for manipulating the activity of 

key signaling nodes or pathways and are often used for dissecting the signaling pathways that regulate 

basic cellular functions or underlie disease. Small molecule chemical inhibitors are useful for achieving 

pathway inhibition, and the choice of inhibitor depends on the objective of the study. Some chemical 

inhibitors act at the receptor level, blocking all signaling emanating from a single ligand receptor complex 

and thus inhibiting multiple pathways at once. For example, the tyrosine kinase inhibitor gefitinib 

inhibits EGFR and prevents signaling through the MAPK, Akt, and many other pathways activated by 

the receptor. Other chemical inhibitors target a single pathway by inhibiting a key node within the cascade 

such as inhibition of the MAPK pathway using the MEK1 inhibitor PD184352 or U0126. Chemical 

inhibitors are used in a broad range of applications; however, recent research has largely focused on 

understanding their roles in the complex signaling networks of cancer and other disease states such as 

diabetes. Many of these agents are now in clinical development as candidate therapeutics. 

The ability to activate a pathway can be equally important. This can be achieved using chemical activators 

such as AICAR, which activates the AMPK pathway, or by treating cells with various growth factors 

or cytokines that are signaling receptor ligands. 

mLST8 

SIN1 

mTOR 

FKBP12 

GβL 

Rictor 

PRAS40 

Thr246 P 

Rapamycin 

P 

Tyr373 Tyr376 

Ser241 P P P PIP3 

PDK1 

PIP 3 

P Akt/PKB 

Thr308 

P 

Ser473 

Ser2481 

P 

GβL 

Raptor 

mTOR 

PTEN 

Ser939 P 

Rheb 

TSC1 

p85 

GF-R 

PI3K Grb 

IRS 

P 

TSC2 

P 

P P 

Ser1254 

Thr1462 

Tyr1571 

Sos Ras 

Ser2448 

P 

Ser371 

Thr229 

P P 

S6K P Thr421 

Thr389 P P Ser424 

mLST8 

Ser259 

Ser236 

Ser235 

P P P Ser240 

P Ser244 

S6 

cytoplasm 

P Ser259 

Raf P Ser338 

Ser217 

P 

MEK1/2 

P 

Ser221 

Thr202 P P Tyr204 

MAPK/ERK1/2 

PD98059 

(MEK1) 

U0126 

Cytokines and 


Cytokines and Growth Factors from 

CST are produced and bio-assayed 

in-house, ensuring the highest 

standards of quality and consistency. 

www.cellsignal.com/ck&gf 

Chemical modulators, cytokines, 

and growth factors can be used to: 

• Study the role of specific signaling nodes in disease 

• Inhibit a signaling pathway to investigate signaling networks or study activation 

of alternative pathways (e.g. cancer resistance mechanisms) 

• Activate or inhibit an individual signaling node to identify kinase substrates or protein interactions 

• Study immune response or cellular differentiation associated with a particular cytokine 

For example, the chemical modulator crizotinib was used to inhibit activity of the receptor tyrosine 

kinase ALK in KARPAS-299 cells. As shown in the western blot below, phosphorylation of ALK at 

Tyr1604, which is a readout for ALK activity, was completely inhibited by treating cells with 1000 nM 

crizotinib. Lower doses were less effective, with 100 nM crizotinib resulting in partial inhibition of ALK 

activity, while phospho-ALK levels in the 10 nM and 1 nM crizotinib-treated samples were similar to 

the untreated control. 

The receptor tyrosine kinase 

ALK is inhibited by crizotinib. 

Crizotinib #4401: WB analysis of extracts from KARPAS-299 cells, untreated 

or treated with #4401 (1 hr) at the indicated concentrations, using Phospho-ALK 

(Tyr1604) Antibody #3341 (upper) or ALK (D5F3 ® ) XP ® Rabbit mAb #3633 (lower). 

kDa 

140 

100 

80 

60 

50 

40 

140 

100 

80 

60 

50 

40 

0 

1000 

100 

10 

1 

Phospho-ALK 

(Tyr1604) 

ALK (D5F3) 

Crizotinib (nM) 

Commonly Used Chemical Modulators 

Gene Expression, Epigenetics, Nuclear Function 

#2112 Cycloheximide Blocks protein biosynthesis by inhibiting protein elongation 

#5927 Doxorubicin Inhibits DNA and RNA synthesis by intercalating the DNA helix; inhibits topoisomerase I 

#2200 Etoposide Inhibits topoisomerase II resulting in DNA breakage; induces apoptosis 

#9676 Leptomycin B Inhibits nuclear export activity of XPO1/exportin 1 

#9950 Trichostatin A (TSA) Inhibits class I and class II histone deactylases 

Cell Growth and Death 

Cell Growth and Translation 

RTKs 

#4401 Crizotinib Inhibitor of ALK and ROS1 (ATP-competitive inhibitor) 

#5083 Erlotinib Inhibits EGFR (ATP-competitive inhibitor) 

#4765 Gefitinib Inhibits EGFR (active site inhibitor) 

#9084 Imatinib Inhibits Bcr-Abl, PDGFR, c-Kit (active site inhibitor) 

#12121 Lapatinib Inhibits EGFR and HER2 (ATP-competitive inhibitor) 


#12328 Sunitinib Broad RTK inhibitor (PDGFR, VEGFR, c-KIT, RET, CSF-1R, FLT-3/CD135) 

#9842 Tyrphostin AG 1478 Inhibits EGFR 

#12998 Vatalanib Broad RTK inhibitor (VEGFR, PDGFR-B, c-KIT) 

MAP Kinase 

#2222 Anisomycin Inhibits protein synthesis; activates stress-activated kinases (SAPK/JNK, p38-MAPK) 

#11916 Chelerythrine Chloride Cell-permeable inhibitor of PKC; Activates SAPK/JNK and p38-MAPK; induces apoptosis in 

some cell lines 

#12147 PD184352 Highly selective, noncompetitive inhibitor of MEK1 and MEK2 

#9900 PD98059 Highly selective inhibitor of MEK1 and MEK2; binds inactive forms and prevent activation by 

upstream kinases 

#9903 U0126 Highly selective inhibitor of MEK1 and MEK2 

#8158 SB202190 Cell-permeable, highly specific inhibitor of p38-MAPK (ATP-competitive inhibitor) 

#5633 SB203580 Cell-permeable inhibitor of p38-MAPK (inhibits PDK1 at higher concentrations) 


#8177 SP600125 Cell-permeable, highly specific inhibitor of JNK-family kinases 

PD184352 inhibits 

MEK1/2 and blocks 

signaling through the 

MAPK pathway. 

kDa 

100 

80 

60 

50 

40 

30 

20 

100 

80 

60 

50 

40 

30 

20 

– + + 

– 

– 

2 

+ + 

0.2 

0.02 

Phospho-p44/42 

MAPK (Erk1/2) 

(Thr202/Tyr204) 

p44/42 MAPK 

(Erk1/2) 

TPA 

PD184352 (µM) 

PD184352 #12147: WB analysis of 

extracts from HeLa cells, serum-starved 

overnight and untreated or treated with 

TPA #4174 (200 nM, 20 min) either with 

or without #12147 pretreatment (1 hr) 

at the indicated concentrations, using 

Phospho-p44/42 MAPK (Erk1/2) (Thr202/ 

Tyr204) (D13.14.4E) XP ® Rabbit mAb 

#4370 (upper) or p44/42 MAPK (Erk1/2) 

(137F5) Rabbit mAb #4695 (lower). 


www.cellsignal.com/modulation 

267


PI3 Kinase/Akt Pathway 

#12017 Everolimus Specific inhibitor of mTORC1 protein complex 

#9901 LY294002 Reversible but potent inhibitor of PI3K family members 

#9904 Rapamycin Specific inhibitor of mTORC1 protein complex 

#9951 Wortmannin Irreversible, potent inhibitor of PI3K faimly members 

Other Kinases and Phosphatases 

#9902 Calyculin A Cell permeable, selective inhibitor of PP1 and PP2A 

#9052 Dasatinib Tyrosine kinase inhibitor (Abl, Bcr/Abl, Src, c-KIT, Ephrin receptors) 

#9084 Imatinib Tyrosine kinase inhibitor (Abl, Bcr/Abl, c-KIT, PDGFR) 

#12209 Nilotinib Tyrosine kinase inhibitor (Abl, Bcr/Abl, c-KIT, LCK, Ephrin receptors, DDR1/2, PDGFR-B) 

#5934 Okadaic Acid Cell permeable, selective inhibitor of PP1, PP2A, and PP2B 

#9493 PKC412 Very broad spectrum protein kinase inhibitor 

(conventional PKCs, some RTKs, Syk, Cdk1/B, c-Src, etc) 

#9885 Roscovitine Potent and selective inhibitor of CDK1, 2, and 5 (ATP-competitive) 


#9944 AICAR AMP analog that activates AMPK 

#9996 Oligomycin ATP synthase inhibitor; inhibits oxidative phosphorylation 


#9841 Bisindolylmaleimide I, Potent inhibitor of PKC family members 

Hydrochloride 

#9973 Cyclosporin A Inhibits calcineurin 

#9974 FK-506 Inhibits calcineurin 

#3828 Forskolin Potent activator of adenylate cyclase, used to increase levels of cAMP 

#12060 Gö6976 Potent inhibitor of calcium-dependent PKC family members 

#9844 H-89, Dihydrochloride Broad spectrum Ser/Thr kinase inhibitor used to inhibit PKA; also inhibits the activity of 

p70S6K, MSK, and ROCKII and others 

#9995 Ionomycin, Calcium Salt Calcium ionophore; activates calcium-/calmodulin-dependent 

kinase and calcium-dependent PKCs 

#9953 Staurosporine Very broad, ATP-competitive protein kinase inhbitor (PKC, PKA, Src, CaM kinase, etc.) 

#4174 TPA (12-O-Tetradecanoylphorbol-13-Acetate) 

Cell permeable, potent activator of PKC; also used to activate MAPK 

Cell Biology 

#8132 17-AAG Inhibits HSP90 chaperone activity resulting in degradation of HSP90 client proteins 

#2204 Bortezomib Proteasome inhibitor 

#9972 Brefeldin A Inhibits ER to Golgi protein transport; triggers UPR and apoptosis 

#9886 Docetaxel Inhibits microtubule depolymerization; prevents cell division 

#9843 Geldanamycin Inhibits HSP90 chaperone activity resulting in degradation of HSP90 client proteins 

#2194 MG-132 Proteasome inhibitor 

#2190 Nocodazole Inhibits microtubule polymerization; induces cell cycle arrest in G2/M-phase 

#9807 Paclitaxel Inhibits microtubule depolymerization; prevents cell division 

#12758 Thapsigargin Inhibits ER calcium-ATPases; induces ER stress 

#12819 Tunicamycin Inhibits glycoprotein synthesis and causes G1 cell cycle arrest 

Growth factor activation of signaling 

with IGF-I followed by chemical inhibition 

of mTOR using everolimus 

hIGF-I #8917 and Everolimus #12017: WB analysis of extracts from HeLa 

cells, serum-starved overnight and treated with #8917 (100 ng/ml, 10 min) either 

with or without #12017 pretreatment (1 hr) at the indicated concentrations, using 

Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP ® Rabbit mAb #4858 

(upper) or S6 Ribosomal Protein (5G10) Rabbit mAb #2217 (lower). 


kDa 

100 

80 

60 

50 

40 

30 

20 

100 

80 

60 

50 

40 

30 

20 

– + 

– – 

+ + 

1000 

100 

Phospho-S6 

Ribosomal 

Protein 

(Ser235/236) 

S6 Ribosomal 

Protein 

+ + hIGF-I 

Everolimus (nM) 

10 

1 

23 

Localization & Classification 

using conjugated primary antibodies 

How can I visualize protein location or simultaneously 

analyze intracellular signaling events within cell populations 

Antibody conjugation is the process by which a fluorescent dye, small molecule, enzyme, or solid 

matrix label is attached covalently to an antibody. Fluorescent dye conjugates are routinely used 

for detection and quantification of cell surface and intracellular proteins within cell populations by 

flow cytometry (FC) and visualization of protein location by immunofluorescence (IF). Other antibody 

conjugates, such as those labeled with small molecules, enzymes, or beads are used for detection or 

analysis of proteins within cell lysates by western blot (WB), ELISA, or immunoprecipitation (IP). For all 

labels, the key benefit of conjugated primary antibodies is the ability to directly detect target proteins, 

thereby eliminating the need for a secondary antibody. 

Conjugated primary antibodies can be used to: 

• Save time by eliminating the secondary antibody incubation and wash steps 

• Eliminate potential background attributable to secondary antibody 

• Multiplex using primary antibodies from the same host species 

• Perform high throughput screening with one-step staining protocol 

Conjugation Types 

Labels Examples Applications 

Fluorescent dyes Alexa Fluor ® , Pacific Blue , PE, FITC, APC Flow, IF 

Small molecule Biotin WB, ELISA 

Enzyme HRP, alkaline phosphatase WB, ELISA, dot blot 

Solid matrix Sepharose ® or magnetic beads IP, cell sorting 

Conjugation Chemistry 

The conjugation process commonly occurs by one of two methods. First, a label can be covalently 

attached to an antibody at primary amine groups on exposed lysine residues within the immunoglobulin 

protein. Although this conjugation method is highly effective, the exact location and density of label can 

change from antibody to antibody and potentially for each labeling reaction due to the high number of 

lysine residues within the average antibody molecule. In addition, conjugation of lysine residues in the 

variable region can disrupt antibody-antigen binding. A second method of conjugation occurs at sulfhydryl 

groups from cysteine residues forming the disulfide bonds that stabilize antibody structure. An 

initial chemical reduction step disrupts the bonds and makes these groups available for conjugation. 

Researchers typically target the disulfide bonds within the hinge region for this type of conjugation, 

as the single location within the antibody molecule lends itself to consistent and uniform labeling. The 

optimal conjugation chemistry is unique for each antibody and must be determined experimentally. 

The Importance of Optimization 

Conjugated antibodies must be properly validated in order to generate maximum signal:noise ratio. In 

addition to determining optimal label chemistry, it is also important to consider optimal label density. 

Each conjugation reaction can produce variations in degree of labeling (DOL)—that is, the number of 

label molecules attached to a single antibody. Insufficient DOL will result in a weak signal, whereas 

a higher DOL may create an overall brighter signal with higher background, resulting in lower signal 

to noise. As shown in the graph, antibodies with the highest DOL do not necessarily perform better 

than those antibodies with a lower DOL. Optimized DOL is critical when working with low abundance 

proteins or low affinity antibodies in order to avoid false negative data. 

www.cellsignal.com/localization 269


chapter 23: Localization & classification 

Optimal antibody-to-dye ratio is critical for maximum performance. 

Custom conjugations are optimized by degree of 

labeling testing to identify the optimal antibody: 

dye molecule ratio, resulting in conjugates with 

maximum performance. 

DOL 2.34 

DOL 4.28 

DOL 5.51 

DOL 7.27 

How CST conjugated antibodies can benefit your research: 

• Rigorously Validated: optimal conjugation chemistry and DOL are determined for each antibody 

in their intended application, producing conjugates with maximal performance and eliminating 

additional optimization steps 

• Highly Reproducible Results: extensive testing and rigorous validation protocols minimize 

lot-to-lot variation 

• Assay Flexibility: custom conjugation is available for Alexa Fluor ® 488, 555, 594, or 647 dyes; 

PE; Pacific Blue dye; Sepharose ® or magnetic beads; biotin; and HRP labels 

Fluorescent Dye Antibody Conjugates 

Fluorescent signal is produced when light energy from a laser or metal halide lamp at a specific 

wavelength is absorbed by a fluorochrome (excitation) and then released (emission), producing light at 

distinct wavelengths that can be measured by fluorescence detectors. 

Fluorescent dyes can be used in IF to produce images of cell or tissue structures, and are often used 

to study changes in protein localization and/or expression in response to a cell stimulus. Fluorescent 

dye conjugates can be used in FC to analyze single cell signaling events, and are particularly useful 

for multiplex assays because multiple primary antibodies conjugated to different fluorochromes can be 

used to identify protein targets within a single sample regardless of host species, greatly increasing 

assay flexibility. 

Fluorescent dye conjugated antibody used 

to visualize α-Synuclein expression 

α-Synuclein (D37A6) XP ® Rabbit mAb (Alexa Fluor ® 488 Conjugate) #5496: 

Confocal IF analysis of normal rat cerebellum using #5496 (green). Blue pseudocolor 


S/N Ratio 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

0 1 2 3 4 5 

Concentration (µg/ml) 

Multiplex analysis: decreased expression of Sox2 and 

Oct-4 pluripotency markers by day 5 in RA-induced NTERA-2 cells 

Sox2 

A B C 

Oct-4A 

Fluorescent dye conjugated antibody used to detect 

etoposide-induced apoptotic cell populations 

Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (Pacific Blue Conjugate) 

#8788: Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated 

(green), using #8788. 

Events 

Cleaved Caspase-3 (Asp175) 

(Pacific Blue Conjugate) 

Small Molecule, Enzyme, and Bead Conjugates 

Conjugation of an antibody to the horseradish peroxidase (HRP) enzyme is a common strategy for 

detecting proteins in cell lysates by WB or ELISA. HRP catalyzes the oxidation of luminescent or chromogenic 

substrates into detectable light or colored products. Small molecule conjugates such as biotin 

take advantage of the natural affinity between biotin and streptavidin and can be detected directly by 

streptavidin-HRP. Conjugating an antibody to a Sepharose ® or magnetic bead allows for the physical 

separation of the detected protein from a whole cell lysate by centrifugation or magnetic force. Bead 

conjugates can be used to enrich low abundance proteins or to study protein-protein interactions using 

an antibody to a target of interest and analyzing proteins that co-precipitate. 

HRP enzyme labeled antibody 

used for direct WB detection 

kDa 

80 

60 

50 

40 

30 

20 

1 2 3 4 

Cytochrome c (D18C7) Rabbit 

mAb (HRP Conjugate) #12959: 

WB analysis of extracts from various 

cell lines using #12959. 

Lanes 

1. HeLa 3. C2C12 

2. NIH/3T3 4. C6 

Oct-4A (C30A3) Rabbit mAb 

(Alexa Fluor ® 488 Conjugate) 

#5177: Flow cytometric analysis of 

NTERA-2 cells, treated with 10 μM 

retinoic acid (RA) for 5 days to induce 

neuronal differentiation, using #5177 

and Sox2 (D6D9) XP ® Rabbit mAb 

(Alexa Fluor ® 647 Conjugate) #5067. 

At 0 days (A), 3 days (B), and 5 days 

(C) of treatment, cells were harvested, 

fixed, and permeabilized according to 

the CST standard flow protocol and 

analyzed on a 4-laser Gallios Flow 

Cytometer (Beckman Coulter). 

10 

Cytochrome c 

Bead conjugates are used for protein enrichment by IP. 

Fluorescent dye 

conjugated antibody 

used to detect 

nuclear induction 

of phospho-p53. 

Phospho-p53 (Ser15) (16G8) Mouse 


#8695: Confocal IF analysis of HT-29 

cells, untreated (left) or UV-treated 

(right), using #8695 (blue pseudocolor). 

Actin filaments were labeled with 

DyLight 554 Phalloidin #13054 (red). 

Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb (Sepharose ® Bead Conjugate) 

#4074: IP of HeLa cell lysates, untreated or treated with Human Interferon-α1 (hIFN-α1) 

#8927, using XP ® Rabbit (DA1E) IgG (Sepharose ® Bead Conjugate) #3423 and #4074. 

The WB was probed using Phospho-Stat3 (Tyr705) (3E2) Mouse mAb #9138. 

Lanes 

1. Rabbit (DA1E) mAb IgG XP ® Isotype Control 

(Sepharose ® Bead Conjugate) #3423 

2. Phospho-Stat3 (Tyr705) (D3A7) XP ® Rabbit mAb 

(Sepharose ® Bead Conjugate) #4074 

kDa 

200 

140 

100 

80 

60 

50 

40 

30 

1 

2 

– + – + 

Phospho- 

Stat3 

(Tyr705) 

hIFN-α1 


www.cellsignal.com/localization 

271

24 


Screening & Quantification 

using ELISA to screen and quantify 

cellular responses at the target level 

How can I quantify levels of total and post-translationally 

modified proteins in response to cell stimuli 

The sandwich enzyme-linked immunosorbent assay (ELISA) is a quantitative, microplate-based assay 

that detects changes in abundance of a target protein within a cell sample using a capture antibody 

coated to the plate and a detection antibody to generate signal. Unlike a western blot that achieves 

specificity by using a single antibody combined with separation of protein by size, an ELISA is highly 

specific because it uses two antibodies recognizing different epitopes of the same protein. ELISAs are 

broadly utilized in both research and clinical settings; here, we focus on their application in research for 

the study of intracellular signaling using phospho-specific and total protein antibodies. ELISAs can also 

be used to quantify epigenetic changes using histone acetylation- or methylation-specific antibodies. 

The quantitative nature, high sensitivity, plate-based format, and ease-of-use make ELISAs a commonly 

used tool for screening in drug discovery and development. 

Sandwich ELISAs can be used to: 

• Screen therapeutic compounds for effects on post-translational modification of target proteins 

• Measure relative changes in total or post-translationally modified protein levels in response 

to an external stimuli such as a therapeutic compound 

• Assess effects of a cell stimulus at multiple time points or dosages 

• Quantitatively measure activation of signaling proteins in a high throughput format 

• Confirm data generated by western blot or other antibody-based applications 

For example, the PathScan ® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit from Cell Signaling Technology 

(CST) was used to assess phosphorylation of Bcl-2 at Ser70 in Jurkat cells treated with a single dose 

of paclitaxel. As shown in the figure, paclitaxel treatment increases phosphorylation of Bcl-2 at Ser70 

more than 2-fold relative to untreated control without affecting the level of total Bcl-2 protein. The 

changes quantified by ELISA were independently observed by western blot. 

Assessing the effects of growth factor or cytokine stimulation using ELISA 

PathScan ® Phospho-Akt (Thr308) Chemiluminescent 

Sandwich ELISA Kit #7135: Relationship between protein 

concentration of lysates from untreated and PDGF-treated 

NIH/3T3 cells and immediate light generation with chemiluminescent 

substrate is shown. Cells (80% confluence) 

were treated with PDGF #9909 (50 ng/ml) and lysed after 

incubation at 37ºC for 5 min. Graph inset corresponding to 

the shaded area shows high sensitivity and a linear response 

at the low protein concentration range. 

PDGF-treated 

Untreated 

RLU (x10 5 ) 

60 

50 

40 

30 

20 

10 

0 

0.0 

PathScan ® Phospho-Stat3 (Tyr705) Sandwich ELISA Kit #7300: Treatment 

of HeLa cells with IFN-α stimulates phosphorylation of Stat3 at Tyr705, detected 

by #7300, but does not affect the level of total Stat3 protein, detected by a Stat3 

antibody via western analysis. OD 450 readings are shown in the top figure, while 

the corresponding western blot using Phospho-Stat3 (Tyr705) (3E2) Monoclonal 

Antibody #9138 or Stat3 antibody, is shown in the figure to the right. 

Untreated 

IFN-α-treated 

70 

0.1 

0.2 0.3 0.4 0.5 0.6 



4 

3 

2 

1 

0 

14 

10 

6 

2 

Phospho-Stat3 

(Tyr705) 

chapter 24: screening & Quantification 

0.02 0.06 0.1 

0.7 0.8 

Phospho- 

Stat3 

(Tyr705) 

Stat3 

– + IFN-α 

How PathScan ® Sandwich ELISA Kits can benefit your research: 

• Highly Sensitive: detect low abundance target proteins and generate measurable linear 

responses at low lysate concentrations 

• Rigorously Validated Antibody Pairs: generate accurate data and minimize background, 

ensuring reliable results 

• Multiple Detection Options: colorimetric or chemiluminescent detection options are available 

• Specialized Packaging: bulk orders and custom 384-plate size are available 

Paclitaxel treatment of Jurkat cells results 

in phosphorylation of Bcl-2 at Ser70. 

High sensitivity ELISAs produce a broad dose response 

and are linear in low concentration ranges. 

PathScan ® Phospho-Bcl-2 (Ser70) Sandwich ELISA Kit #11874: 

Treatment of Jurkat cells with Paclitaxel stimulates phosphorylation of Bcl-2 

at Ser70, as detected by #11874, but does not affect the levels of total Bcl-2 

detected by PathScan ® Total Bcl-2 Sandwich ELISA Kit #12030. Jurkat cells 

were untreated or treated with λ phosphatase or Paclitaxel #9807 (1 mM, 

20 hr, 37°C). The absorbance readings at 450 nm are shown in the top 

figure, while the corresponding western blots using Phospho-Bcl-2 (Ser70) 

(5H2) Rabbit mAb #2827 (left panel) or Bcl-2 (D55G8) Rabbit mAb (Human 

Specific) #4223 (right panel) are shown in the bottom figure. 

λ phosphatase-treated 

Untreated 

Paclitaxel-treated 


4 

3 

2 

1 

0 

Phospho-Bcl-2 

(Ser70) 

Total Bcl-2 

PathScan ® Phospho-EGF Receptor (Tyr1068) Sandwich ELISA 

Kit #7240: The relationship between protein concentration of lysates 

from untreated and EGF-treated A-431 cells and kit assay optical 

density readings. After starvation, A-431 cells (85% confluence) were 

treated with EGF #8916 (100 ng/ml, 5 min, 37°C) and then lysed. 

EGF-treated 

Control 

2.5 

1.5 

1.0 

0.5 

Absorbance 450nm 2.0 

0 

0.05 0.10 0.15 0.20 0.25 


0.30 

– – + 

+ – – 

– – + 

+ – – 

Paclitaxel 

λ Phosphatase 

In a second example, the PathScan ® Phospho-Akt (Thr308) Chemiluminescent Sandwich ELISA Kit 

#7135 was used to examine the effects growth factor stimulation on phosphorylation of Akt at Thr308, 

an indicator of Akt activation and signaling. As shown in the figure, PDGF-mediated activation of Akt 

was quantified along a range of lysate levels, demonstrating the kit’s high sensitivity and a linear 

response at low protein concentrations. A similar assessment of the effects of cytokine stimulation on 

phosphorylation of Stat3 at Tyr705 was performed using #7300. 






www.cellsignal.com/screening 

273

25 

Cell 


Monitoring 

using cell assays to monitor the health of 

cells and their response to multiple challenges 

How can you monitor cell state or cellular response 

to a specific treatment within a population of cells 

Cell assays are a diverse set of research tools used to assess cell functions such as growth, apoptosis, 

or signaling at the cellular level. Cell-based assays are commonly used in a broad array of biological 

disciplines. In general, these assays are highly sensitive and easy to perform. Many cell-based assays are 

conducted in 96-well plate format, allowing the researcher to process a large number of samples quickly. 

Cell assays can be used to: 

• Perform large-scale screening of therapeutic compounds or other stimuli 

• Evaluate cell response to multiple drug dosages at various time points 

• Complement data obtained by antibody-based applications 

• Determine if treatment data obtained through other experiments correlates 

to cell proliferation, senescence, or death 

Proliferation and Viability Assays 

Cell proliferation and viability assays allow a researcher to ask basic questions about cell status or 

responses to physiological stimuli or challenges without having to focus on the underlying molecular 

mechanism or individual protein target. These assays have broad applications in both basic research and 

drug discovery. Measuring cell proliferation and viability is often the first step in examining the effects 

of an unknown compound on cell health. These assays can also be used to investigate the effects of 

known growth factors or cytotoxic agents in combination with a test compound, or on cells containing 

genetic mutations or knockdowns within key signaling nodes. 


• BrdU Assays measure cell proliferation by treating cells with a pyrimidine analog, BrdU, which 

incorporates into the DNA of replicating cells and can be measured using a BrdU antibody. 

• XTT and Resazurin Assays measure cell viability by colorimetric or fluorescent detection of 

reduced tetrazolium salt, an indicator of living cells. 

Doxorubicin-treated cells are viable but do not proliferate. 

4.0 

3.0 

2.0 

1.0 

XTT 

BrdU 

% Inhibition 

10 

0 

-3 -2 -1 0 1 2 -10 -2 -1 0 1 2 

110 

90 

70 

50 

30 

Log (Doxorubicin, µM) 

XTT Cell Viability Kit #9095 and BrdU Cell Proliferation Assay Kit #6813: C2C12 cells were seeded at 2x10 4 cells/well in a 96-well 

plate and incubated overnight. Cells were then treated with various concentrations of doxorubicin overnight. The cytotoxicity was measured 

using #9095 (red) and #6813 (blue) as shown in the left panel. The percentage inhibition in each assay was calculated and plotted in the 

right panel. Doxorubicin treatment can lead to cell DNA damage followed by cell cycle arrest. 

Death Assays 

Identifying agents that induce or prevent cell death is critical for studies in cancer, cardiovascular 

research, and other pathologies. 

• The Annexin V-FITC Early Apoptosis Detection Assay differentiates between early and 

late apoptosis using flow cytometry; annexin V binding to phosphatidylserine indicates early 

apoptosis, whereas late apoptotic cells will have lost plasma membrane integrity, allowing 

propidium iodide to passively enter the cell. 

• The Caspase-3 Activity Assay monitors caspase-3 activity as a readout for apoptosis by 

measuring cleavage of a fluorescent caspase-3 substrate. 

Camptothecin-treated 

cells undergo apoptosis. 

Annexin V-FITC Early Apoptosis 

Detection Kit #6592: Flow cytometric 

analysis of Jurkat cells untreated (left) or 

treated with camptothecin (10µM, 4 hr; 

right) using #6592. 

Propidium Iodide 

Late Apoptotic/ 

Necrotic Cells 

Live Cells 

Untreated 

Early Apoptotic Cells 

Annexin V-FITC 

Late Apoptotic/ 

Necrotic Cells 

Live Cells 

Camptothecin-treated 

Early Apoptotic Cells 

Second Messenger Assays 

Second messenger assays allow a researcher to evaluate signaling mechanisms at work in a cell 

population by assessing levels of cyclic adenosine 3’, 5’ monophosphate (cAMP) and cyclic guanosine 

3’, 5’ monophosphate (cGMP). cAMP and cGMP are involved in numerous cell signaling pathways, 

and alterations in concentration of these molecules are associated with several disease states such as 

cardiovascular disease, neuronal disorders, and cancer. cAMP and cGMP are generated through the 

activity of adenylyl cyclase (AC) or guanylyl cyclase (GC), respectively, and are degraded by phosphodiesterases 

(PDEs). 

• cAMP and cGMP Assays can be used to infer enzymatic activity of AC/GC or PDEs by 

measuring levels of cAMP or cGMP through a competition enzyme-linked immunoassay. 

A cAMP assay is a competition ELISA used to 

measure cAMP levels in forskolin-treated CHO cells. 


4.0 

3.0 

2.0 

0.5mM IBMX 

No IBMX 

% Activity 

30 

1.0 

EC 50: 

10 

0.58 µM (No IBMX) 

0.09 µM (0.5 mM IBMX) 

0 

-3 -2 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3 

110 

90 

70 

50 

Log (Forskolin, µM) 

Cyclic AMP XP ® Assay Kit #4339: Treatment of CHO cells with Forskolin #3828 increases cAMP concentration as detected by #4339. 

CHO cells were seeded at 4x10 4 cells/well in a 96-well plate and incubated overnight. Cells were either left untreated or pretreated with 

0.5 mM IBMX for 30 min prior to forskolin treatment (15 min) and lysed with 1X Cell Lysis Buffer #9803. The absorbance values (left) and 

percentage of activity (right) are shown above. 

The percentage of activity is calculated as follows: % activity=100x[(A-A basal )/(A max -A basal )], where A is the sample absorbance, A max 

is the absorbance at maximum stimulation (i.e., high forskolin concentration), and A basal is the absorbance at basal level (no forskolin). 

Forskolin directly activates adenylyl cyclases and increases cellular cAMP concentration. IBMX is a non-specific inhibitor of cAMP and 

cGMP phosphodiesterases and promotes accumulation of cAMP and cGMP in cells. 

chapter 25: Monitoring 

BrdU, XTT and 

Resazurin Assay Kits 

#6813 BrdU Cell Proliferation 

Assay Kit 

#5492 BrdU Cell Proliferation 

Chemiluminescent Assay Kit 

#11884 Resazurin Cell Viability Kit 

#9095 XTT Cell Viability Kit 

#9860 Senescence β-Galactosidase 

Staining Kit 

Annexin V-FITC and 

Caspase-3 Assay Kits 

#6592 Annexin V-FITC Early 

Apoptosis Detection Kit 

#5723 Caspase-3 Activity Assay Kit 

cAMP and cGMP Assay Kits 

#4339 Cyclic AMP XP ® Assay Kit 

#8019 Cyclic AMP XP ® 


#4360 Cyclic GMP XP ® Assay Kit 

#8020 Cyclic GMP XP ® 


GTPase Detection Kits 

#8816 Active Arf1 Detection Kit 

#8819 Active Cdc42 Detection Kit 

#8815 Active Rac1 Detection Kit 

#8818 Active Rap1 Detection Kit 

#8821 Active Ras Detection Kit 

#8820 Active Rho Detection Kit 


www.cellsignal.com/monitoring 275


Small GTPase Detection Assays 

Like second messenger assays, small GTPase detection assays allow a researcher to evaluate cell 

signaling by measuring the presence of activated Ras GTPase as a readout for signaling activity. The 

Ras GTPase superfamily consists of five protein families (Ras, Rho, Rab, Arf, and Ran) whose members 

cycle between the active, GTP-bound, and inactive, GDP-bound, state. GTPases play an important role 

in cell cycle progression, cell survival, and actin cytoskeletal organization and are actively studied in 

many fields, including tumor biology. 

• GTPase Detection Assays measure activation of a single GTPase in the cell using a GST-fusion 

protein to bind the activated form of GTP-bound Rac1 (or other GTPase), which can then be 

immunoprecipitated with glutathione resin and analyzed by western blot. 

GTPase detection assays assess levels 

of active GTPase in a cell lysate. 

Active Rac1 Detection Kit #8815: NIH/3T3 cell lysates (500 µl at 1 mg/ml) were treated in vitro 

with GTPγS or GDP to activate or inactivate Rac1 (refer to optional step C in protocol). The lysates 

were then incubated with glutathione resin and GST-PAK1-PBD (lanes 2 and 3). GTPγS-treated 

lysate was also incubated without GST-PAK1-PBD in the presence of glutathione resin as a 

negative control (lane 4). WB analysis of cell lysate (20 µg, lane 1) or 20 µl of the eluted samples 

(lanes 2–4) was performed using a Rac1 Mouse mAb. Anti-mouse IgG, HRP-linked Antibody 

#7076 was used as the secondary antibody. 

1 2 3 4 

– + – – GDP 

– – + + GTPγS 

– + + – GST-PAK1-PBD 

26 

Verification 

using siRNA-mediated knockdown to verify function 

How can I ask a highly targeted question about protein 

or antibody function in the context of the whole cell 

Small interfering RNAs (siRNA) are short segments (19–27 nucleotides) of double stranded RNA that 

silence expression of a single gene in a sequence-specific manner. This is achieved when a single 

strand of the RNA duplex associates with the RNA-induced silencing complex (RISC), a multiprotein 

complex containing endonucleases that bind and degrade mRNA at a sequence complementary to the 

siRNA. Thus, transfection of a specific siRNA molecule into living cells can result in specific inhibition of 

expression of the corresponding protein. 

siRNA can be used to: 

• Verify an antibody is detecting its intended target 

• Analyze the functional role of a node within a signaling pathway 

• Validate a potential therapeutic target 

• Explore gene function in the context of a disease or biological system 

The GTP-bound 

GTPase pull-down 

process can be 

divided into 3 steps. 

Step 1: Mix sample, binding protein, 

and glutathione resin in the spin cup 

and incubate at 4ºC to allow GTP-bound 

GTPase binding to the glutathione resin 

through GST-linked binding protein. 

Step 2: Remove unbound proteins by 

centrifugation. 

Step 3: Elute glutathione resin-bound 

GTPase with SDS buffer. The eluted 

sample can then be analyzed by WB. 

sample 

spin cup 

collection tube 

= Other Proteins 

= GTP-bound GTPase 

= GDP-bound GTPase 

= Binding Protein 

= Glutathione 

Step 1 Step 2 

Step 3 

- mix sample with resin 

and binding protein 

- incubate 

wash 

elute 

Western blot 

For example, in the experiment below, SignalSilence ® p27 Kip1 siRNA from Cell Signaling Technology is 

used to verify antibody specificity. The robust signal obtained from p27 Kip1 (D69C12) XP ® Rabbit mAb 

#3686 in the presence of control siRNA (-) is diminished when p27 Kip1 expression is silenced using 

two different p27 Kip1 siRNAs (+), indicating the antibody specifically detects the p27 Kip1 protein. 

siRNA-mediated knockdown 

verifies p27 Kip1 

antibody specificity. 

kDa 

80 

60 

50 

40 

30 

20 

50 

40 

I 

II 

– + + 

p27 

Kip1 

β-Actin 

p27 Kip1 

siRNA 

SignalSilence ® p27 Kip1 siRNA I #12324 

and siRNA II #12410: WB analysis of extracts 

from HeLa cells, transfected with 100 nM 

SignalSilence ® Control siRNA (Unconjugated) 

#6568 (-), #12324 (+), or #12410 (+), using p27 

Kip1 (D69C12) XP ® Rabbit mAb #3686 (upper) 

or β-Actin (D6A8) Rabbit mAb #8457 (lower). The 

p27 Kip1 (D69C12) XP ® Rabbit mAb confirms 

silencing of p27 Kip1 expression, while the β-Actin 

(D6A8) Rabbit mAb is used as a loading control. 

SignalSilence ® siRNA 

Experimental Controls 

SignalSilence ® Control siRNA 

(Fluorescein Conjugate) #6201 

Transfection efficiency 

SignalSilence ® Control siRNA 

(Unconjugated) #6568 

siRNA specificity 

How SignalSilence ® siRNA can benefit your research: 

• Two siRNAs Available for Most Targets: confirm inhibition at the functional protein 

level using two equally potent sequences 

• Rigorously Validated Duplexes: confidently inhibit expression of your intended protein 

• Appropriate Controls: monitor transfection efficiency with a fluorescein-labeled 

nontargeted siRNA and specificity with an unconjugated control 

• Target Proteins From Different Species: inhibit expression of human or mouse targets 

A positive control 

siRNA confirms c-Myc 

siRNA specificity in the 

target cells, suggesting 

that absence of c-Myc 

expression is due to 

specific knockdown. 

SignalSilence ® Control 

siRNA (Unconjugated) #6568: 

Confocal IF analysis of HeLa 

cells, transfected with #6568 

(left) or SignalSilence ® c-Myc 

siRNA I #6341 (right), using 

c-Myc (D84C12) XP ® Rabbit mAb 

#5605 (green). Actin filaments 

were labeled with DyLight 554 

Phalloidin #13054 (red). 


www.cellsignal.com/verification 277

27 


Customization 

antibodies in a carrier-free 

formulation for specific assay platforms 

How can I obtain an antibody formulated for 

the special requirements of my assay platform 

Researchers studying protein activities and associations can choose from a wide array of high throughput 

assay platforms. Many of these require antibodies in specific concentrations or in formulations free 

of BSA or other carrier proteins that are typically found in off-the-shelf antibody storage buffers. Custom 

antibody formulations from Cell Signaling Technology (CST) are highly specific, in-house validated 

antibodies that are ready to be labeled or used directly in your assay platform, providing the flexibility 

necessary for your unique experiment. 

Single peak indicates the antibody is intact and shows no cleavage or degradation. 

AU 

0.070 

0.065 

0.060 

0.055 

0.050 

0.045 

0.040 

0.035 

0.030 

0.025 

0.020 

Single peak indicates that the 

antibody is intact and shows 

no cleavage or degradation. 

chapter 27: Customization 

PD-L1 (E1L3N ® ) XP ® Rabbit mAb 

#13684: Size exclusion chromatogram 

of #13684 formulated in PBS buffer. 

Custom formulated antibodies can be used to: 

• Label antibodies with fluorochromes, lanthanides, or molecules for use on specific platforms such 

as AlphaScreen ® , DELFIA ® or HTRF assays 

• Develop pair-based sandwich assays for use on specific platforms such as Bio-Plex ® or Luminex ® 

• Develop custom plate-based sandwich ELISA kits 

• Design custom multiplex assays where several protein readouts are assayed using multiple targetspecific 

antibodies 

0.015 

0.010 

0.005 

0.000 

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 

minutes 

CST can supply antibodies in phosphate buffered saline (PBS) or borate buffer at your specified concentration. 

Once the antibodies are formulated in the specified buffer, we validate them for molecular integrity 

by size exclusion chromatography and application-specific functionality using WB, IP, peptide ELISA, 

flow cytometry, IHC, or IF. The application testing is dependent on the proposed use of the antibody. 

Peptide ELISA confirms phospho-specificity of multiple antibody lots. 

How custom formulated antibodies 

from CST can benefit your research: 

• CST is ISO9001:2008 certified for the production and supply of monoclonal antibodies, ensuring 

reproducible, traceable product quality 

• Antibodies are supplied in formulations adaptable to multiple platforms 

• Each antibody is custom manufactured with complete documentation support to ensure 

product integrity 

• Dedicated custom antibody personnel actively manage the supply chain for full transparency from 

initial enquiries to production and post-sales collaborative support. Key features of the supply chain 

process include: 

– Specific lots of antibody material can be reserved for long-term multiple purchases 

– Custom packaging ensures that the material is available in an experiment-or 

platform-ready format 

– CST freezer-to-customer traceability ensures peace of mind and guarantees product integrity 

and performance 

– Priority Alert Plus shipping with SenseAware SM technology from FedEx minimizes product transit 

time and allows continuous monitoring of temperature during shipment 

– Scientists specializing in unique application support are available to support custom 

product clients 

• All catalog antibodies are available in bulk volumes, which ensures long term supply. 

Lot reservation is available to reserve a single lot of any of our reagents. 

Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb #9167: Peptide ELISA analysis of multiple lots of #9167, using plates 

coated with phospho-peptides (Tyr-P) or non-phospho-peptides (NP). The data shows minimal lot-to-lot variation. 


CST Custom Formulated Antibodies 

CST’s primary antibodies are available in a carrier free formulation at custom concentrations that allow antibody 

labeling, conjugation or custom assays. Visit our website to learn more. www.cellsignal.com/custom 

www.cellsignal.com/customization 

279

IV 

Additional 

Information 


About CST 

Nature Conservancy 

Publications by CST Scientists 

Index 

Diagram & Table Keys 

Contact Information 

280 

Adhesion 

Depiction of cytoskeletal and extracellular structures and proteins involved in adhesion, including 

an adherens junction and integrin-mediated cellular interaction with the extracellular matrix. 


281

28 

Section IV: Additional Information 

About CST 

Behind the Antibodies 

The preceding sections in this book offer proof that we are serious about both advancing science and 

the quality of our products. But what about the company and people behind the antibodies If you met 

someone from CST, you would learn that we are scientists ourselves, we value the respect of our peers, 

are very proud of the work we do, and are just as interested as the people who buy our products in the 

outcome of their research. Like everyone else, we have our own back-story about who we are, how we 

came to be where we are now, and our dreams for the future. We’d like to introduce you to that CST. 

Science is our passion, and it’s how we pay the bills—we create and sell antibodies and related 

products that can be used in research aspiring to improve human health. But we are also citizens of our 

local and global communities. We are parents who care about the quality of science taught at our local 

schools, accountants who care about the environment, business leaders concerned with the footprint 

we leave behind, and scientists curious about art. 

As a private company we have the freedom to do things differently. We can invest our profits in our 

own translational research programs. We can focus on long-term goals without having to answer to 

the stock market each quarter. But we don’t just throw money around and hope to make a difference. 

We hire people who are very good at what they do and disciplined enough to do it both in and out of 

the lab. We have several volunteer committees within CST that independently determine how to spend 

funds we have set aside for various initiatives. It is what sets us apart from many other businesses. 

Corporate Social Responsibility 

Corporate Donations Committee 

As a company, CST sets aside money to help local organizations share in our success and find additional 

funding for selected initiatives. How that money is spent is determined by our employees. Our Corporate 

Donations Committee (CDC) is a group of enthusiastic volunteers at our US locations who decide on their 

own where that money can do the most good. Twice a year the CDC reviews requests and distributes 

funds to local schools, non-profit organizations, and charities. 

It’s a special culture where the responsibility of granting money is handed to employees. Involving 

employees in some of CST’s philanthropy helps develop the kind of culture that makes a difference and 

empowers them to feel part of the CST mission. 

Education in Science 

Naturally, we think that science education is important to keep discovery alive in the future. Because 

most of us can think of a teacher or an experience that sparked our interest in science, CST’s Education 

in Science program supports local public high schools by giving teachers access to much needed 

grant money for resources that keep science fun and engaging. 

At the college level, our Summer Internship Program provides interns from local colleges and communities 

with a positive work experience alongside our associates within the lab and outside of the 

lab. It’s gratifying (and fun) to see some of these interns return as full-time employees after completing 

their degree. 

The Arts 

Exchanging ideas in a supportive environment fosters the kind of creativity that both scientists and 

artists thrive in. Creative people are appreciated at CST, and the three gallery spaces in our two US 

facilities exhibit the creativity of some of New England’s finest artists. Our artist receptions and the discussions 

about their work always bring a different perspective in viewing artwork. A short walk through 

our labs and office spaces reveals walls filled with the work of artists that have previously exhibited at 

CST and which continue to enrich anyone who cares to stop and have a closer look. 

You can see why we think CST is a special kind of company. We are about more than making money. 

We are trying to build a business that’s not only great for the customers, but also rewarding for our 

employees and appreciated as a member of our local and global communities. 

We hire smart people with a commitment and passion to succeed and make a difference. These are 

the people behind the antibodies and every product we make. 

Environmental Commitment 

As a global citizen, CST recognizes that our business has real impact on the planet. We are working to 

lower our carbon footprint and limit the impact we make on the environment. In addition to incorporating 

sustainability into our own operations, CST also works within our communities to help restore 

ecosystems by supporting environmental organizations working in the field to promote conservation. 

This has been part of our mission since CST was founded in 1999. 

Sustainability 

We’re not forced into being good stewards of the environment. We’re doing it because we care. 

For example: 

Transportation Management Plan 

Our Transportation Management Plan (TMP) uses financial incentives and up to two days off work to 

promote alternative transportation such as carpooling, walking, biking, or taking public transportation. 

This has helped engaged and concerned employees lower our carbon emissions from commuting by 

nearly 8% in less than two years. 

Responsible Marketing and Packaging 

Since 2009, our Massachusetts locations have been shipping our products using a new shipping 

cooler composed from cardboard, mineral wool, rock wool, slag wool, and biodegradable plastic. This 

new shipping cooler has the same thermal properties as a Styrofoam box but can degrade in a landfill. 

Employee Community Garden Program 

CST’s community garden program provides fresh summer produce for the employees and our in-house 

cafeterias at our two Massachusetts locations. In the Netherlands, our employees enjoy fresh locally 

sourced fruit. 

The examples above are only a few examples of how we are working to protect our planet. Integral to 

all of these sustainability efforts is CST’s employee run Green Committee. The vision of this committee 

is to understand the company’s environmental impacts, create awareness within the organization, 

and help develop strategies to minimize environmental impact. This committee continues to find 

sustainable business solutions and develops successful, award-winning programs that have made a 

difference in our industry and our communities. 

Environmental Awareness and Support 

Moving science forward by helping solve important questions about disease and cancer is our mission 

but ultimately, human health depends on the health of our planet. It would be misguided to cure 

ourselves of disease, only to find we had nowhere to live, not enough food to eat or no clean air to 

breathe. Understanding the interactions that shape the living planet on a global level is just as important 

to us as the complex molecular interactions involved in cellular signaling. 

Each year for the CST Nature Calendar, we select a specific environmental theme that highlights not 

only the extraordinary beauty of nature, but also touches upon an important environmental issue 

affecting life on our planet. 

chapter 28: About CST 

“At CST we measure 

our performance in 

the world by practicing 

triple bottom line 

economics: People, 

Planet, and Profit.” 

David Comb, Director of 

Corporate Social Responsibility 

Produce from CST’s Employee Garden 

Atrium at CST Headquarters, Danvers, MA 

Trees Red; Estelle Disch 

282 For Research Use Only. Not For Use in Diagnostic Procedures. 

www.cellsignal.com/cstcommitments 

283

29 CST Nature Conservancy 


Sagono 

Bamboo Forest 

This small patch of bamboo forest, 

on the outskirts of Kyoto, is a popular 

Japanese tourist destination that 

people have visited for thousands 

of years because of its sense of 

peacefulness and tranquility. 

Conservancy Efforts & Resources 

Human know-how still cannot replace nature, but we have acquired the means of disrupting natural 

processes on a global scale. With this newfound ability comes a responsibility to manage the planet’s 

resources wisely. By safeguarding vital ecosystems whose services we depend on, rather than plundering 

them for immediate gain, we can ensure that nature flourishes. In so doing, humans will benefit too. 

As part of CST’s commitment to the environment, each year we support non-profit organizations that are 

helping to protect the earth’s magnificent biodiversity and the ecosystems that sustain life on our planet. 

chapter 29: CST Nature Conservancy 

2015 

Protected Landscapes 

The best way to protect endangered 

species is often to protect the land these 

creatures call home. Usually, the bigger 

the swath of land that can be set aside, 

the better. The same general approach 

holds for preserving treasured forests, 

mountains, waterfalls, rivers, and lakes. 

Ecosystems blend into each other, and 

nature, accordingly, does not abide 

by the artificial boundaries drawn by 

humans—even though natural systems 

can, of course, be heavily impacted by 

societal decisions and activities. 

Support: 

• Essex County Greenbelt Association 

• The Jurassic Coast Trust 

• Kunming Institute of Botany 

• Wild Bird Society of Japan 

Jurassic Coast on the eastern shore of England. © Joaquin Pinho/Getty Images 

Red Crowned Crane is 

an endangered species 

found only in Japan 

and China. Famous for 

their complex dancing 

rituals, this rare species 

is most threatened by 

habitat destruction. 

© Kerstin Hinze/Minden Pictures 

2014–13 

Magnificent Marine 

Environments 

Images of Earth by astronauts nearly 

four decades ago dramatically proved 

what we knew but only dimly appreciated—the 

Earth is a water planet. But 

the sea-blue dominating those images 

concealed as much as it revealed. Unlike 

the land’s sculpted landscape, which 

suggested diversity and begged for 

further exploration, the uniform surface 

of the ocean suggested sameness. This 

could not be further from the truth as we 

continue to find startling new life forms 

and are constantly reminded just how 

dependent we are on the ocean. 

As coastal populations and economic 

activity continue to grow, the need for 

protecting additional coastal areas will 

grow as well. 

Support: 

• Southern Environmental Association 

• Toledo Institute for Development 

and Environment 

• Ecologic Development Fund 

• Large Pelagics Research Center 

• Oceana 

• Conservation Law Foundation 

• Ipswich River Watershed Association 

• Nature Conservancy 

• New England Aquarium 

© Akira Kaede/Getty Images 


Spinyhead Blenny (Acanthemblemaria spinosa) emerges from its coral home to feed at Lighthouse Reef, Belize. 

© Brian J. Skerry/National Geographic Creative 

www.cellsignal.com/cstcsr 

285



2013 

Mesoamerican Reef 

Extending more than 600 miles from 

Isla Contoy, a bird sanctuary near the 

Yucatan Peninsula, to the Bay Islands 

of Honduras, the Mesoamerican Reef is 

the largest barrier reef in the Western 

Hemisphere and the second largest 

in the world. About one-third of this 

natural wonder, and arguably the choicest 

stretch of reef, lies off the coast of 

Belize. Charles Darwin once described 

this complex—consisting of dazzling 

corals, crystal clear blue waters, and a 

host of aquatic denizens—as “the most 

remarkable reef in the West Indies.” 

Support: 

• Southern Environmental Association 

• Toledo Institute for Development 

and Environment 



2009–10 

Conservation 

Without Borders 

Nature does not stop at the borders 

of a national park or between nations. 

Appreciation of that fact has led to a 

growing realization among conservationists 

that the best, and perhaps only, 

way to preserve the environment 

and protect wildlife is through large 

landscapes that transcend the artificial 

boundaries drawn by humans. Animals 

need room to rove for any number of 

reasons—and these needs cannot 

be met within isolated sanctuaries 

standing as islands amidst a sea of 

development. The home range for a 

single grizzly bear, for example, can 

cover 1,000 square miles—and wolves 

roam even farther. Securing movement 

corridors and other “linkages” between 

designated wilderness areas has thus 

become a cornerstone of an emerging 

conservation strategy. 

Support: 

• Yellowstone to Yukon 

• Intl. Gorilla Conservation 


• Gombe School of Environment 

and Society 

Cushion sea star (Oreaster reticulatus) sitting in sea grass beds (Thalassia), South Water Caye, Belize. © Tony Rath/tonyrath.com 

Eastern Gorillas, Volcanoes National Park, Rwanda. © Thomas Marent/Minden Pictures 

2011–12 

Conservation 

Treasures 

Since its founding in 1951, The Nature 

Conservancy (TNC) has protected about 

120 million acres in all 50 American 

states and more than 30 countries. In 

that time, the organization has focused 

its conservation efforts on what it calls 

“The Last Great Places”—habitats 

deemed special in terms of the plant 

and animal species they harbor, as well 

as the natural processes they sustain. 

Support: 

• Appalachian Mountain Club 


• Essex County Greenbelt Association 

• Gombe School of Environment 

and Society 



• The Nature Conservancy 

• Trout Unlimited, Alaska 

2007–08 

World’s Fisheries 

in Crisis 

For most of human history, it seemed 

inconceivable that our species could 

make an appreciable dent in the ocean’s 

bounty. Surely we’d do nothing to 

jeopardize the principal source of animal 

protein for a billion people. Yet today, with 

millions of fishing vessels patrolling the 

seas and marine life decimated in their 

wake, ocean observers know the opposite 

to be true: “In all the seas there are very 

few places left with undisturbed fish 

stocks”, claims Boris Worm of Dalhousie 

University in Halifax, Canada. “The whole 

ocean has been transformed.” 

Support: 

• New England Aquarium 

• Conservation Law Foundation 

Atlantic Salmon (Salmo salar), 

St. Jean River, Quebec 

Giant sequoia (Sequoiadendron giganteum) in Yosemite National Park, California. © David Noton/Minden Pictures 

Leafy sea dragon (Phycodurus eques), found only in temperate waters around southern 

and western Australia. © Paul Sutherland/National Geographic Creative 

286 For Research Use Only. Not For Use in Diagnostic Procedures. www.cellsignal.com/cstcsr 

© Paul Nicklen 

287



2005–06 

Spectacular 

Migrations 

Each year, traveling by land, air, and 

sea, animals make time-honored 

journeys in pursuit of their destiny. They 

follow paths taken by their forebears, 

obeying cues they alone perceive and 

dare not ignore. Swimming, flying, running, 

and crawling, by day and by night, 

these creatures venture forth in large 

groups or solo. If movement is life, ours 

is truly a planet in motion. 

Support: 

• Operation Migration 

• The Atlantic Salmon Federation 

2002 

Freshwater Wetlands 

The occupants of planet Earth are 

dependent on an elaborate life-support 

system that maintains the air we breathe, 

regulates temperature, supplies reserves 

of food and water, and shields us from 

deadly radiation. This system, provided 

by nature free-of-charge, offers a broad 

array of critical services: purifying the 

air and water, maintaining soil fertility, 

decomposing and detoxifying wastes, 

recycling essential nutrients, stabilizing 

the climate, protecting us from the sun’s 

ultraviolet rays, mitigating floods and 

droughts, pollinating our crops, and 

controlling agricultural pests. 

Support: 


• Massachusetts Chapter of 

The Nature Conservancy 

Group of caribou (Rangifer tarandus) swimming through an Alaskan river during migration. © Michio Hoshino/Minden Pictures 

Underwater landscape of water lilies, Okavango Delta, Botswana. © Frans Lanting/Frans Lanting Stock 

2003–04 

Plight of the 

Pollinators 

Ecologists are nowhere close to 

documenting all the valuable services 

performed by the 100,000 or more 

species of pollinators. While dispensing 

pollen, bees, bats, birds, and other 

animals also add to ecosystem productivity 

by facilitating the spread of seeds 

and the redistribution of nitrogen-rich 

wastes. With their contributions largely 

unrecognized, pollinators hold their 

place as the unsung heroes of the 

natural world. 

Support: 

• Xerces Society 

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30 


Through their ongoing 

commitment to innovative 

research, CST scientists 

have contributed to a 

wealth of peer-reviewed 

publications in cancer 

research, proteomicsbased 

discovery, and 

novel antibody technology. 

2014 

Gray, V.E. et al. (2014) Signatures of natural selection 

on mutations of residues with multiple posttranslational 

modifications. Mol. Biol. Evol. 31, 1641–1645. 

Oslund, R.C. et al. (2014) A Phosphohistidine 

Proteomics Strategy Based on Elucidation of a Unique 

Gas-phase Phosphopeptide Fragmentation Mechanism. 

J. Am. Chem. Soc. [Epub ahead of print]. 

Mulherkar, S. et al. (2014) The small GTPases RhoA 

and Rac1 regulate cerebellar development by controlling 

cell morphogenesis, migration and foliation. Dev. 

Biol. [Epub ahead of print]. 

2013 

Miller, R.A. et al. (2013) Biguanides suppress hepatic 

glucagon signalling by decreasing production of cyclic 

AMP. Nature 494, 256–260. 

Ling, H. et al. (2013) Transforming growth factor β 

neutralization ameliorates pre-existing hepatic fibrosis 

and reduces cholangiocarcinoma in thioacetamidetreated 

rats. PLoS ONE 8, e54499. 

Mulherkar, S. et al. (2013) The Small GTPase RhoA Is 

Required for Proper Locomotor Circuit Assembly. PLoS 

ONE 8, e67015. 

Israelsen, W.J. et al. (2013) PKM2 Isoform-Specific 

Deletion Reveals a Differential Requirement for Pyruvate 

Kinase in Tumor Cells. Cell 155, 397–409. 

Hitosugi, T. et al. (2013) Tyr26 phosphorylation of 

PGAM1 provides a metabolic advantage to tumours 

by stabilizing the active conformation. Nat. Commun. 

4, 1790. 

Morandell, S. et al. (2013) A Reversible Gene-Targeting 

Strategy Identifies Synthetic Lethal Interactions 

between MK2 and p53 in the DNA Damage Response 

In Vivo. Cell Rep. 5, 868–877. 

2012 

Hornbeck, P.V. et al. (2012) PhosphoSitePlus: a 

comprehensive resource for investigating the structure 

and function of experimentally determined posttranslational 

modifications in man and mouse. Nucleic 

Acids Res. 40, 261–270. 

Katayama, R. et al. (2012) Mechanisms of acquired 

crizotinib resistance in ALK-rearranged lung Cancers. 

Sci. Transl. Med. 4, 120ra17. 

Feng, H. et al. (2012) Phosphorylation of dedicator 

of cytokinesis 1 (Dock180) at tyrosine residue 

Y722 by Src family kinases mediates EGFRvIII-driven 

glioblastoma tumorigenesis. Proc. Natl. Acad. Sci. USA 

109, 3018–3023. 

Garnett, M.J. et al. (2012) Systematic identification of 

genomic markers of drug sensitivity in cancer cells. 

Nature 483, 570–575. 

Cheung, W.C. et al. (2012) A proteomics approach for 

the identification and cloning of monoclonal antibodies 

from serum. Nat. Biotechnol. 30, 447–452. 


Publications 

by CST Scientists 

Stokes, M.P. et al. (2012) PTMScan direct: identification 

and quantification of peptides from critical signaling 

proteins by immunoaffinity enrichment coupled 

with LC-MS/MS. Mol. Cell Proteomics 11, 187–201. 

Ren, H. et al. (2012) Identification of anaplastic 

lymphoma kinase as a potential therapeutic target in 

ovarian cancer. Cancer Res. 72, 3312–3323. 

Rimkunas, V.M. et al. (2012) Analysis of Receptor 

Tyrosine Kinase ROS1-Positive Tumors in Non-Small 

Cell Lung Cancer: Identification of a FIG-ROS1 Fusion. 

Clin. Cancer Res. 18, 4449–4457. 

Xu, W. et al. (2012) Dynamic tyrosine phosphorylation 

modulates cycling of the HSP90-P50(CDC37)-AHA1 

chaperone machine. Mol. Cell 47, 434–443. 

Hardee, M.E. et al. (2012) Resistance of glioblastomainitiating 

cells to radiation mediated by the tumor 

microenvironment can be abolished by inhibiting transforming 

growth factor-β. Cancer Res. 72, 4119–4129. 

Hezel, A.F. et al. (2012) TGF-β and αvβ6 integrin act 

in a common pathway to suppress pancreatic cancer 

progression. Cancer Res. 72, 4840–4845. 

Hitosugi, T. et al. (2012) Phosphoglycerate mutase 1 

coordinates glycolysis and biosynthesis to promote 

tumor growth. Cancer Cell 22, 585–600. 

Sato, S. et al. (2012) Proteomics-directed cloning of 

circulating antiviral human monoclonal antibodies. 


Buckley, S.M. et al. (2012) Regulation of pluripotency 

and cellular reprogramming by the ubiquitin-proteasome 

system. Cell Stem Cell 11, 783–798. 

Stokes, M.P. et al. (2012) Quantitative Profiling of DNA 

Damage and Apoptotic Pathways in UV Damaged Cells 

Using PTMScan Direct. Int J. Mol. Sci. 14, 286–307. 

Foulkes-Murzycki, J.E. et al. (2012) Cooperative Effects 

of Drug-Resistance Mutations in the Flap Region 

of HIV-1 Protease. ACS Chem. Biol. 8, 513–518. 

Li, Z. et al. (2012) Gr-1+CD11b+ cells are responsible 

for tumor promoting effect of TGF-β in breast cancer 

progression. Int. J. Cancer 131, 2584–2595. 

2011 

Hülper, P. et al. (2011) Tumor localization of an 

anti-TGF-β antibody and its effects on gliomas. 

Int. J. Oncol. 38, 51–59. 

Gu, T.L. et al. (2011) Survey of tyrosine kinase signaling 

reveals ROS kinase fusions in human cholangiocarcinoma. 

PLoS ONE 6, e15640. 

Kettenbach, A.N. et al. (2011) Absolute quantification 

of protein and post-translational modification abundance 

with stable isotope-labeled synthetic peptides. 

Nat. Protoc. 6, 175–186. 

Verstraeten, V.L. et al. (2011) Protein farnesylation 

inhibitors cause donut-shaped cell nuclei attributable 

to a centrosome separation defect. Proc. Natl. Acad. 

Sci. USA 108, 4997–5002. 

Dammer, E.B. et al. (2011) Polyubiquitin linkage 

profiles in three models of proteolytic stress suggest 

the etiology of Alzheimer disease. J. Biol. Chem. 286, 

10457–10465. 

Brave, S.R. et al. (2011) Assessing the activity of cediranib, 

a VEGFR-2/3 tyrosine kinase inhibitor, against 

VEGFR-1 and members of the structurally related 

PDGFR family. Mol. Cancer Ther. 10, 861–873. 

Pighi, C. et al. (2011) Phospho-proteomic analysis of 

mantle cell lymphoma cells suggests a pro-survival 

role of B-cell receptor signaling. Cell. Oncol. (Dordr) 

34, 141–153. 

Gu, T.L. et al. (2011) Survey of activated FLT3 

signaling in leukemia. PLoS ONE 6, e19169. 

Yadav, H. et al. (2011) Protection from obesity and 

diabetes by blockade of TGF-β/Smad3 signaling. 

Cell Metab. 14, 67–79. 

Kremer, K.N. et al. (2011) Stromal Cell-Derived 

Factor-1 Signaling via the CXCR4-TCR Heterodimer 

Requires Phospholipase C-{beta}3 and Phospholipase 

C-{gamma}1 for Distinct Cellular Responses. 

J. Immunol. 187, 1440–1447. 

Zhang, Q. et al. (2011) TGF-β regulates DNA methyltransferase 

expression in prostate cancer, correlates 

with aggressive capabilities, and predicts disease 

recurrence. PLoS ONE 6, e25168. 

Kim, W. et al. (2011) Systematic and quantitative 

assessment of the ubiquitin-modified proteome. 

Mol. Cell 44, 325–340. 

Emanuele, M.J. et al. (2011) Global Identification of 

Modular Cullin-RING Ligase Substrates. Cell 147, 

459–474. 

Feng, H. et al. (2011) Activation of Rac1 by Src-dependent 

phosphorylation of Dock180Y1811 mediates 

PDGFRα-stimulated glioma tumorigenesis in mice and 

humans. J. Clin. Invest. 121, 4670–4684. 

Biswas, S. et al. (2011) Anti-transforming growth 

factor ß antibody treatment rescues bone loss and 

prevents breast cancer metastasis to bone. PLoS ONE 

6, e27090. 

Bouquet, F. et al. (2011) TGFβ1 inhibition increases 

the radiosensitivity of breast cancer cells in vitro and 

promotes tumor control by radiation in vivo. Clin. 

Cancer Res. 17, 6754–6765. 

Fan, J. et al. (2011) Tyrosine Phosphorylation of 

Lactate Dehydrogenase A Is Important for NADH/ 

NAD+ Redox Homeostasis in Cancer Cells. Mol. Cell. 

Biol. 31, 4938–4950. 

Hitosugi, T. et al. (2011) Tyrosine phosphorylation 

of mitochondrial pyruvate dehydrogenase kinase 1 

is important for cancer metabolism. Mol. Cell 44, 

864–877. 

Lee, K.A. et al. (2011) Ubiquitin ligase substrate 

identification through quantitative proteomics at both 

the protein and peptide levels. J. Biol. Chem. 286, 

41530–41538. 

Lonning, S. et al. (2011) Antibody targeting of TGF-β 

in cancer patients. Curr. Pharm. Biotechnol. 12, 

2176–2189. 

2010 

Moritz, A. et al. (2010) Akt-RSK-S6 kinase signaling 

networks activated by oncogenic receptor tyrosine 

kinases. Sci. Signal. 3, ra64. 

van Vugt, M.A. et al. (2010) A mitotic phosphorylation 

feedback network connects Cdk1, Plk1, 53BP1, and 

Chk2 to inactivate the G(2)/M DNA damage checkpoint. 

PLoS Biol. 8, e1000287. 

Gu, T.L. et al. (2010) Identification of activated 

Tnk1 kinase in Hodgkin’s lymphoma. Leukemia 24, 

861–865. 

Pecquet, C. et al. (2010) Induction of myeloproliferative 

disorder and myelofibrosis by thrombopoietin receptor 

W515 mutants is mediated by cytosolic tyrosine 112 

of the receptor. Blood 115, 1037–1048. 

Nishimura, E.K. et al. (2010) Key roles for transforming 

growth factor beta in melanocyte stem cell maintenance. 

Cell Stem Cell 6, 130–140. 

Hudon, V. et al. (2010) Renal tumour suppressor 

function of the Birt-Hogg-Dubé syndrome gene 

product folliculin. J. Med. Genet. 47, 182–189. 

Hirschey, M.D. et al. (2010) SIRT3 regulates mitochondrial 

fatty-acid oxidation by reversible enzyme 

deacetylation. Nature 464, 121–125. 

Schoeberl, B. et al. (2010) An ErbB3 antibody, 

MM-121, is active in cancers with ligand-dependent 

activation. Cancer Res. 70, 2485–2494. 

Ghosh, R. et al. (2010) Phellodendron amurense 

bark extract prevents progression of prostate tumors 

in transgenic adenocarcinoma of mouse prostate: 

potential for prostate cancer management. Anticancer 

Res. 30, 857–865. 

Kang, S. et al. (2010) p90 ribosomal S6 kinase 2 

promotes invasion and metastasis of human head and 

neck squamous cell carcinoma cells. J. Clin. Invest. 

120, 1165–1177. 

Ganapathy, V. et al. (2010) Targeting the Transforming 

Growth Factor-beta pathway inhibits human basal-like 

breast cancer metastasis. Mol. Cancer 9, 122. 

Takaku, S. et al. (2010) Blockade of TGF-beta 

enhances tumor vaccine efficacy mediated by CD8(+) 

T cells. Int. J. Cancer 126, 1666–1674. 

Bonnette, P.C. et al. (2010) Phosphoproteomic characterization 

of PYK2 signaling pathways involved in 

osteogenesis. J. Proteomics 73, 1306–1320. 

Aguiar, M. et al. (2010) Gas-Phase Rearrangements 

Do Not Affect Site Localization Reliability in 

Phosphoproteomics Data Sets. J. Proteome Res. 9, 

3103–3107. 

Carretero, J. et al. (2010) Integrative genomic and 

proteomic analyses identify targets for Lkb1-deficient 

metastatic lung tumors. Cancer Cell 17, 547–559. 

Andersen, J.N. et al. (2010) Pathway-based identification 

of biomarkers for targeted therapeutics: personalized 

oncology with PI3K pathway inhibitors. Sci. Transl. 

Med. 2, 43ra55. 

Buel, G.R. et al. (2010) Fyn promotes phosphorylation 

of collapsin response mediator protein 1 at tyrosine 

504, a novel, isoform-specific regulatory site. J. Cell 

Biochem. 111, 20–28. 

Knowlton, M.L. et al. (2010) Profiling Y561-dependent 

and -independent substrates of CSF-1R in epithelial 

cells. PLoS ONE 5, e13587. 

Edwards, J.R. et al. (2010) Inhibition of TGF-β 

signaling by 1D11 antibody treatment increases bone 

mass and quality in vivo. J. Bone Miner. Res. 25, 

2419–2426. 

chapter 30: publications by cst scientists 

www.cellsignal.com/cstpubs 291


chapter 30: publications by cst scientists 

Publications by 

CST Scientists 

Please visit our website to view 

publications prior to 2003. 

www.cellsignal.com/cstpubs 

Heo, J.M. et al. (2010) A stress-responsive system 

for mitochondrial protein degradation. Mol. Cell 40, 

465–480. 

Demir, E. et al. (2010) The BioPAX community 

standard for pathway data sharing. Nat. Biotechnol. 

28, 935–942. 

Bennett, E.J. et al. (2010) Dynamics of cullin-RING 

ubiquitin ligase network revealed by systematic quantitative 

proteomics. Cell 143, 951–965. 

2009 

Vitsky, A. et al. (2009) Homeostatic role of transforming 

growth factor-beta in the oral cavity and esophagus 

of mice and its expression by mast cells in these 

tissues. Am. J. Pathol. 174, 2137–2149. 

Terabe, M. et al. (2009) Synergistic enhancement 

of CD8+ T cell-mediated tumor vaccine efficacy by 

an anti-transforming growth factor-beta monoclonal 

antibody. Clin. Cancer Res. 15, 6560–6569. 

Ueda, R. et al. (2009) Systemic inhibition of transforming 

growth factor-beta in glioma-bearing mice 

improves the therapeutic efficacy of glioma-associated 

antigen peptide vaccines. Clin. Cancer Res. 15, 

6551–6559. 

Gordus, A. et al. (2009) Linear combinations of docking 

affinities explain quantitative differences in RTK 

signaling. Mol. Syst. Biol. 5, 235. 

Hitosugi, T. et al. (2009) Tyrosine phosphorylation inhibits 

PKM2 to promote the Warburg effect and tumor 

growth. Sci. Signal. 2, ra73. 

Harrington, M.G. et al. (2009) The morphology and 

biochemistry of nanostructures provide evidence for 

synthesis and signaling functions in human cerebrospinal 

fluid. Cerebrospinal Fluid Res. 6, 10. 

Pan, S. et al. (2009) Mass spectrometry based targeted 

protein quantification: methods and applications. 

J. Proteome Res. 8, 787–797. 

Boccalatte, F.E. et al. (2009) The enzymatic activity 

of 5-aminoimidazole-4-carboxamide ribonucleotide 

formyltransferase/IMP cyclohydrolase is enhanced by 

NPM-ALK: new insights in ALK-mediated pathogenesis 

and the treatment of ALCL. Blood 113, 2776–2790. 

Kang, S. et al. (2009) Fibroblast growth factor receptor 

3 associates with and tyrosine phosphorylates 

p90 RSK2, leading to RSK2 activation that mediates 

hematopoietic transformation. Mol. Cell. Biol. 29, 

2105–2117. 

ten Klooster, J.P. et al. (2009) Mst4 and Ezrin induce 

brush borders downstream of the Lkb1/Strad/Mo25 

polarization complex. Dev. Cell 16, 551–562. 

Xu, P. et al. (2009) Quantitative proteomics reveals 

the function of unconventional ubiquitin chains in 

proteasomal degradation. Cell 137, 133–145. 

Yu, J. et al. (2009) Mutation-specific antibodies for 

the detection of EGFR mutations in non-small-cell lung 

cancer. Clin. Cancer Res. 15, 3023–3028. 

Haack, H. et al. (2009) Diagnosis of NUT midline 

carcinoma using a NUT-specific monoclonal antibody. 

Am. J. Surg. Pathol. 33, 984–991. 

Yu, Y. et al. (2009) A site-specific, multiplexed kinase 

activity assay using stable-isotope dilution and highresolution 

mass spectrometry. Proc. Natl. Acad. Sci. 

USA 106, 11606–11611. 


Kasyapa, C. et al. (2009) Phosphorylation of the 

SSBP2 and ABL proteins by the ZNF198-FGFR1 fusion 

kinase seen in atypical myeloproliferative disorders as 

revealed by phosphopeptide-specific MS. Proteomics 

9, 3979–3988. 

Schwer, B. et al. (2009) Calorie restriction alters mitochondrial 

protein acetylation. Aging Cell 8, 604–606. 

Kubota, K. et al. (2009) Sensitive multiplexed analysis 

of kinase activities and activity-based kinase identification. 


Ji, H. et al. (2009) EGF-induced ERK activation promotes 

CK2-mediated disassociation of alpha-Catenin 

from beta-Catenin and transactivation of beta-Catenin. 

Mol. Cell 36, 547–559. 

Lechtreck, K.F. et al. (2009) The Chlamydomonas 

reinhardtii BBSome is an IFT cargo required for export 

of specific signaling proteins from flagella. J. Cell Biol. 

187, 1117–1132. 

2008 

Fattouh, R. et al. (2008) Transforming growth factorbeta 

regulates house dust mite-induced allergic airway 

inflammation but not airway remodeling. Am. J. Respir. 

Crit. Care Med. 177, 593–603. 

Perry, K. et al. (2008) Treatment of transforming 

growth factor-beta-insensitive mouse Renca tumor by 

transforming growth factor-beta elimination. Urology 

72, 225–229. 

Nam, J.S. et al. (2008) An anti-transforming growth 

factor beta antibody suppresses metastasis via cooperative 

effects on multiple cell compartments. Cancer 

Res. 68, 3835–3843. 

Nam, J.S. et al. (2008) Transforming growth factor 

beta subverts the immune system into directly promoting 

tumor growth through interleukin-17. Cancer Res. 

68, 3915–3923. 

Frantz, S. et al. (2008) Transforming growth factor 

beta inhibition increases mortality and left ventricular 

dilatation after myocardial infarction. Basic Res. 

Cardiol. 103, 485–492. 

Guo, A. et al. (2008) Signaling networks assembled by 

oncogenic EGFR and c-Met. Proc. Natl. Acad. Sci. USA 

105, 692–697. 

Pan, S. et al. (2008) Application of targeted quantitative 

proteomics analysis in human cerebrospinal fluid using 

a liquid chromatography matrix-assisted laser desorption/ionization 

time-of-flight tandem mass spectrometer 

(LC MALDI TOF/TOF) platform. J. Proteome Res. 7, 

720–730. 

Kang, S. et al. (2008) Epidermal growth factor 

stimulates RSK2 activation through activation of 

the MEK/ERK pathway and src-dependent tyrosine 

phosphorylation of RSK2 at Tyr-529. J. Biol. Chem. 

283, 4652–4657. 

Li, K. et al. (2008) Regulation of WRN protein cellular 

localization and enzymatic activities by SIRT1-mediated 

deacetylation. J. Biol. Chem. 283, 7590–7598. 

Dentin, R. et al. (2008) Hepatic glucose sensing 

via the CREB coactivator CRTC2. Science 319, 

1402–1405. 

Taylor, E.B. et al. (2008) Discovery of TBC1D1 as an 

insulin-, AICAR-, and contraction-stimulated signaling 

nexus in mouse skeletal muscle. J. Biol. Chem. 283, 

9787–9796. 

Miyake, Z. et al. (2007) Activation of MTK1/MEKK4 

by GADD45 through induced N-C dissociation and 

dimerization-mediated trans autophosphorylation 

of the MTK1 kinase domain. Mol. Cell. Biol. 27, 

2765–2776. 

Li, Y., et al. (2008) HDAC6 is required for epidermal 

growth factor-induced beta-catenin nuclear localization. 

J. Biol. Chem. 283, 12686–12690. 

Kaushansky, A. et al. (2008) A quantitative study of 

the recruitment potential of all intracellular tyrosine 

residues on EGFR, FGFR1 and IGF1R. Mol. Biosyst. 4, 

643–653. 

Kaushansky, A. et al. (2008) System-wide investigation 

of ErbB4 reveals 19 sites of Tyr phosphorylation that 

are unusually selective in their recruitment properties. 

Chem. Biol. 15, 808–817. 

Pan, W. et al. (2008) Wnt3a-mediated formation of 

phosphatidylinositol 4,5-bisphosphate regulates LRP6 

phosphorylation. Science 321, 1350–1353. 

Altarejos, J.Y. et al. (2008) The Creb1 coactivator 

Crtc1 is required for energy balance and fertility. Nat. 

Med. 14, 1112–1117. 

Stratford, A.L. et al. (2008) Y-box binding protein-1 

serine 102 is a downstream target of p90 ribosomal 

S6 kinase in basal-like breast cancer cells. Breast 

Cancer Res. 10, R99. 

Stokes, M.P. et al. (2007) Profiling of UV-induced ATM/ 

ATR signaling pathways. Proc. Natl. Acad. Sci. USA 

104, 19855–19860. 

Engelman, J.A. et al. (2008) Effective use of PI3K 

and MEK inhibitors to treat mutant Kras G12D and 

PIK3CA H1047R murine lung cancers. Nat. Med. 14, 

1351–1356. 

2007 

Liu, V.C. et al. (2007) Tumor evasion of the immune 

system by converting CD4+CD25- T cells into 

CD4+CD25+ T regulatory cells: role of tumor-derived 

TGF-beta. J. Immunol. 178, 2883–2892. 

Nakanishi, H. et al. (2007) TGF-beta-neutralizing 

antibodies improve pulmonary alveologenesis and 

vasculogenesis in the injured newborn lung. Am. J. 

Physiol. Lung Cell Mol. Physiol. 293, 151–161. 

Gerber, S.A. et al. (2007) The absolute quantification 

strategy: application to phosphorylation profiling of 

human separase serine 1126. Meth. Mol. Biol. 359, 

71–86. 

Gu, T.L. et al. (2007) Phosphoproteomic analysis 

identifies the M0-91 cell line as a cellular model for 

the study of TEL-TRKC fusion-associated leukemia. 

Leukemia 21, 563–566. 

Li, X. et al. (2007) Akt/PKB regulates hepatic metabolism 

by directly inhibiting PGC-1alpha transcription 

coactivator. Nature 447, 1012–1016. 

Dong, S. et al. (2007) 14-3-3 Integrates prosurvival 

signals mediated by the AKT and MAPK pathways 

in ZNF198-FGFR1-transformed hematopoietic cells. 

Blood 110, 360–369. 

Gu, T.L. et al. (2007) A novel fusion of RBM6 to CSF1R 

in acute megakaryoblastic leukemia. Blood 110, 

323–333. 

Kang, S. et al. (2007) FGFR3 activates RSK2 to mediate 

hematopoietic transformation through tyrosine 

phosphorylation of RSK2 and activation of the MEK/ERK 

pathway. Cancer Cell 12, 201–214. 

Kass, E.M. et al. (2007) Stability of checkpoint kinase 

2 is regulated via phosphorylation at serine 456. 

J. Biol. Chem. 282, 30311–30321. 

Rikova, K. et al. (2007) Global survey of phosphotyrosine 

signaling identifies oncogenic kinases in lung 

cancer. Cell 131, 1190–1203. 

2006 

Nam, J.S. et al. (2006) Bone sialoprotein mediates the 

tumor cell-targeted prometastatic activity of transforming 

growth factor beta in a mouse model of breast 

cancer. Cancer Res. 66, 6327–6335. 

Steen, H. et al. (2006) Phosphorylation analysis by 

mass spectrometry: myths, facts, and the consequences 

for qualitative and quantitative measurements. 

Mol. Cell Proteomics 5, 172–181. 

Shalizi, A. et al. (2006) A calcium-regulated MEF2 

sumoylation switch controls postsynaptic differentiation. 

Science 311, 1012–1017. 

Kling, D.E. et al. (2006) Distribution of ERK1/2 and 

ERK3 during normal rat fetal lung development. Anat. 

Embryol. 211, 139–153. 

Goss, V.L. et al. (2006) A common phosphotyrosine 

signature for the Bcr-Abl kinase. Blood 107, 

4888–4897. 

Cheng, D. et al. (2006) Relative and absolute quantification 

of postsynaptic density proteome isolated from 

rat forebrain and cerebellum. Mol. Cell Proteomics 5, 

1158–1170. 

Kirkpatrick, D.S. et al. (2006) Quantitative analysis of 

in vitro ubiquitinated cyclin B1 reveals complex chain 

topology. Nat. Cell Biol. 8, 700–710. 

Walters, D.K. et al. (2006) Activating alleles of JAK3 

in acute megakaryoblastic leukemia. Cancer Cell 10, 

65–75. 

Griswold, I.J. et al. (2006) Kinase domain mutants of 

Bcr-Abl exhibit altered transformation potency, kinase 

activity, and substrate utilization, irrespective of sensitivity 

to imatinib. Mol. Cell. Biol. 26, 6082–6093. 

Walters, D.K. et al. (2006) Phosphoproteomic analysis 

of AML cell lines identifies leukemic oncogenes. Leuk. 

Res. 30, 1097–1104. 

Mercher, T. et al. (2006) JAK2T875N is a novel 

activating mutation that results in myeloproliferative 

disease with features of megakaryoblastic leukemia in 

a murine bone marrow transplantation model. Blood 

108, 2770–2779. 

Beausoleil, S.A. et al. (2006) A probability-based 

approach for high-throughput protein phosphorylation 

analysis and site localization. Nat. Biotechnol. 24, 

1285–1292. 

Gu, T.L. et al. (2006) Phosphotyrosine profiling identifies 

the KG-1 cell line as a model for the study of 

FGFR1 fusions in acute myeloid leukemia. Blood 108, 

4202–4204. 

www.cellsignal.com/cstpubs 

293

31 


Index 

by Pathways, Protocols, and targets 

Pathway Diagrams and Tables 

Adherens Junction Dynamics 110 

Akt Binding Partners 65 

Akt Substrates 55 

AMPK Signaling 146 

AMPK Substrates 147 

Amyloid Plaque and Neurofibrillary Tangle 190 

Formation in Alzheimer’s Disease 

Angiogenesis Signaling in 

171 

Tumor Neovascularization 

Autophagy Signaling 97 

B Cell Receptor Signaling 178 


84 

Kinase-Disease Associations 

Cell Cycle Control: G1/S Checkpoint 104 

Cell Cycle Control: G2/M DNA Damage 105 

Checkpoint 

Cell Cycle/Checkpoint Control 

102 

Kinase-Disease Associations 

Death Receptor Signaling 92 

Deubiquitinase Table 135 

Dopamine Signaling in Parkinson’s Disease 191 

ErbB/HER Signaling 74 

ESC Pluripotency Differentiation 165 

Examples of Crosstalk Between 

31 

Post-Translational Modifications 

G-Protein Coupled Receptor 

79 

Signaling to MAP Kinase/Erk 

G-Protein Coupled Receptor Signaling: Overview 78 

Hedgehog Signaling 163 

Hippo Signaling 162 

Histone Lysine Methylation 27 

Histone Modifications Table 28 

Inhibition of Apoptosis 91 

Protocols and Troubleshooting Guides 

ChIP General Protocol 196 

ChIP Troubleshooting Guide 200 

Cultured Cells (Immunocytochemistry, IF-IC) 210 

Protocol 

Flow Cytometry Alternate Protocol 204 

Flow Cytometry General Protocol 204 

Frozen/Cryostat Tissue Sections (IF-F) Protocol 211 

IHC Frozen Protocol 

217 

(using SignalStain ® Boost Detection Reagent) 

IHC Paraffin Protocol 

216 

(using SignalStain ® Boost Detection Reagent) 

IHC Troubleshooting Guide 219 

Insulin Receptor Signaling 144 

Jak and Cytokine Receptor Mutants 176 

Jak/Stat Signaling: IL-6 Receptor Family 181 

Jak/Stat Utilization 177 

MAPK/Erk in Growth and Differentiation 46 

Mitochondrial Control of Apoptosis 93 

mTOR Signaling 54 

NF-κB Signaling 182 

Notch Signaling 161 

Nuclear Receptors Signaling 41 

PI3 Kinase/Akt Signaling 53 

Protein Acetylation 26 

Regulation of Actin Dynamics 115 

Regulation of Apoptosis Overview 90 

Regulation of Microtubule Dynamics 116 

SAPK/JNK Signaling 48 

Signaling Pathways Activating p38 MAP Kinase 47 

T Cell Receptor Signaling 179 

TGF-β Signaling 164 

Toll-like Receptor Signaling 180 

Translational Control: Overview 36 

Translational Control: Regulation of elF2 38 

Translational Control: Regulation of elF4E 37 

and p70 S6K 

Tumor Immunology 183 

Tyrosine Kinases Kinase-Disease Associations 71 

Ubiquitin Ligases 125 

Ubiquitin/Proteasome 124 

Vesicle Trafficking in Presynaptic Neurons: 189 

Synchronous Release 

Warburg Effect 145 

Wnt/β-Catenin Signaling 160 

In-Cell Western Protocol 213 

IP Denatured Protein Protocol 237 

IP Native Protein Protocol 235 

Paraffin Tissue Sections (IF-P) Protocol 212 


224 

Antibody Pair Protocol 


223 

Chemiluminescent Protocol 

PathScan ® Sandwich ELISA Colorimetric Protocol 221 

WB Fluorescent Protocol 230 

WB General Protocol 228 

WB Troubleshooting Guide 232 

Targets 

14-3-3 113 

4E-BP 35 

4EHP 35 

4E-T 35 

4F2hc/CD98 142 

5-HTR1A 187 

5-HTR4 187 

53BP1 101 

A1/Bfl-1 88 

A20/TNFAIP3 174 

A2B5 187 

ABCC4 142 

ABCG2 142 

ABIN-1 174 

c-Abl 44 

ACAD 142 

ACAT2 142 

ACE2 119 

AceCS1 142 

Acetyl-CoA Carboxylase 142 

ACF1 24 

Acinus 88 

Ack1 113 

Actin 113 

ACO2 142 

ACPP 69 

ACSL1 142 

α-Actinin 113 

ACVR1 158 

ADAM 109 

ADAMTS1 109, 170 

ADAP 174 

ADAR1 35 

ADH1 142 

Adiponectin 142 

β-Adrenergic Receptor 77, 83 

ADRM1 123 

Afadin 109, 113 

AFP 158 

AGR2 158 

AhR 39 

AID 174 

AIF 88 

AIM2 174 

Aiolos 174 

Ajuba 109 

AKAP 77, 83 

AKR1C2 142 

Akt 51 

ALDH1A1 142 

Aldolase A 142 

Alix 88 

ALK 69 

ALPP 158 

AMACR 142 

Ambra1 96 

AMFR 123 

AML1 174 

AMPA Receptor 187 

AMPK 142 

Amylase 142 

β-Amyloid 187 

Androgen Receptor 39 

Angiopoietin-2 158, 170 

Annexin 83, 113 

ANT2/SLC25A5 142 

AP-2 88 

AP2M1 88, 113 

Apaf-1 88 

APBA2 187 

APC 100, 123, 158 

Ape1 100 

ApoA 83 

ApoE 187 

ApoM 83 

APP/β-Amyloid 187 

APPL1 119 

APS 44 

AQP2 142 

Arf6 119 

Arginase-1 142 

Argonaute 35 

ARID1A/BAF250A 24 

ARK5 142 

Aromatase 39 

ARP 113 

Arrestin 44, 77, 187 

Artemis 100 

AS160 142 

ASCT2 142 

ASF1 24 

ASH2L 24 

ASK1 44 

ASM 83 

Ataxin-1 187 

ATF 44, 142 

Atg 96 

ATGL 142 

Atlastin-1 113 

ATM 100 

ATP Citrate Lyase 142 

ATP2A/SERCA 83 

ATP6V1B1/2 51 

ATPIF1 142 

ATR 100 

ATRIP 100 

AUF1/hnRNP D 35 

Aurora 100 

Aven 88 

Axin 158 

Axl 69 

BACE 187 

BACH 100, 174 

Bad 88 

BAFF 174 

Bag1 119 

BAG6 119 

Bak1 88 

BAP1 123 

BAP31 88 

Basigin/EMMPRIN 174 

Bassoon 187 

BATF 174 

Bax 88 

BCAT 142 

Bcl-2 88 

BCL2L10 88 

BCL6 174 

Bcl10 174 

Bcl-11B 158 

Bcl-w 88 

Bcl-xL 88 

Bcr 44 

Bcr-Abl 44 

Beclin-1 96 

BID 88 

Bif-1 96 

Bik 88 

Bim 88 

BiP 119 

BIRC6 88 

Bit1 88 

Blimp-1/PRDI-BF1 174 

Blk 174 

BLM 100 

BLNK 174 

BMAL 187 

Bmf 88 

Bmi1 24 

BMP 158 

BMPR2 158 

BNIP3 96 

BNIP3L/Nix 96 

Bora 100 

BORIS 24 

Brachyury 158 

BRCA1 100 

BRD 24 

BrdU 100 

chapter 31: Index 

BRE 100 

BRF1/2 35 

Brg1 24 

BRM 24 

Brn2/POU3F2 187 

BRSK 187 

BSP II 109 

BTAF1 24 

Btk 174 

Bub 100 

C1QBP 142 

CA 142, 170 

CABIN1 24 

CACYBP 158 

CAD 142 

Cadherin 109, 170 

Calbindin 187 

Calcineurin A 83 

Caldesmon-1 113 

Calmodulin 83 

Calnexin 119 

Calpastatin 119 

Calumenin 83 

CaMKI 187 

CAND1 123 

CARD 174 

CASK 187 

Caspases 88 

Caspr2 187 

Catalase 142 

Catenin 109, 113, 158 

Cathepsin B 187 

Caveolin-1 113, 119 

CBARA1/MICU1 83 

CBFβ 174 

Cbl-b 123 

c-Cbl 123 

CBP 24, 170 

CCR2 174 

CCT2 119 

CCTα 142 

CD (Cell Surface 

Markers) 

109, 113, 170, 

174, 187 

CD2AP 113 

Cdc2 100 

Cdc6 100 

Cdc7 100 

CDC20 100 

cdc25 100 

CDC37 100, 113, 119 

Cdc42 113 

Cdc45 100 

CDC73 100 


295



CDCP1 158 

CdGAP 113 

CDK 24, 100, 187 

CDT1 100 

CDX2 158 

CEA/CD66e 109 

CEACAM1 158 

C/EBP 142 

CEND1 187 

CENP-A 24 

CENP-T 100 

Centrin-2 113 

CFTR 83 

CHAF1A 24 

CHD 24 

CHFR 100 

β2-Chimerin 113 

CHIP 123 

Chk 100 

Choline Kinase 83 

CHOP 119 

Chronophin/PDXP 113 

CIITA 174 

CIN85 113 

CIRBP 187 

CISH 174 

CK1 100, 158, 187 

Claspin 100 

Clathrin Heavy Chain 119 

Claudin-1 109, 113 

CLCN3 187 

CLIC4 142 

CLIP1/CLIP170 113 

CLK3 35 

CLOCK 24 

CNOT 35 

CNPase 187 

Cofilin 113 

Coilin 35 

Complexin 187 

Connexin 43 109 

Cool/Pix 113 

COPS5 123 

Cortactin 113 

COUP-TF 39 

COX IV 142 

Cox 174 

CP110 100 

CPEB1 35 

C-Peptide 143 

cPLA2 83 

CPT1A 142 

CRABP1 142 

CRADD/RAIDD 88 

CREB 187 

Cripto 158, 170 

CrkII 113 

CrkL 174 

CRMP-2 187 

CRYAB 119 

Csk 44 

CtBP 158 

CTCF 24 

CTDSPL2 24 

CtIP 100 

CTMP 51 

CTR1/SLC31A1 142 

CTR9 24 

CUEDC2 100 

CUL 123 

CXXC1 24 

Cyclic AMP 83 

Cyclin 101 

Cyclin T1 24 

Cyclophilin A 174 

CYLD 123 

CYP3A4 142 

CYP11A1 142 

CYR61 109, 170 

Cytochrome c 88 

Cytokine Receptor 174 

Common β-Chain 

Dab 113, 187 

DAG Lipase 83, 187 

DAP 88 

DAP12 174 

DAPK1 88 

DAPK3/ZIPK 88 

DARPP-32 187 

DAX1 158 

Daxx 88 

DAZL 158 

DBC1 24 

DC-SIGN 174 

DCBLD2 187 

DCP1B 35 

Cleaved Drosophila 88 

Dcp-1 (Asp216) 

DcR 88 

DDB 123 

DDC 187 

DDR 69 

DDX4 35, 158 

Dectin-1 174 

Deltex-2 158 

DEPTOR/DEPDC6 51 

Derlin-1 119 

Desmin 113 

Dexras1 44 

DFF45/DFF35 88 

DGCR8 35 

DHCR24/Seladin-1 142 

DHX29 35 

Diap 113 

Dicer1 35 

DIDO1 89 

DJ-1 51, 187 

DKK 158 

DLK1 158 

DLL 158, 170 

DLST 142 

DMAP1 24 

DNA Polymerase 101 

DNA-PK 101 

DNAJC2/MPP11 119 

DNMT 24 

DOCK180 113 

Dok 44 

Dopamine ß-Hydroxylase 

187 

(DBH) 

Doublecortin 187 

DPYD 142 

DR 24, 89 

DRAK2 89 

Drebrin 187 

Drosha 35 

DRP1 113 

DSG2 113 

DUSP 44 

Dvl 158 

Dynamin 119 

DYRK1 101, 187 

Dysbindin 187 

E2-25K/Hip2 123 

E2A 174 

E2F-1 101 

EAAT 187 

EAPP 101 

EB-1 113 

EDC4/Ge-1 35 

EEA1 119 

eEF 35 

Eg5 101 

EGF Receptor 69 

EGR 187 

eIF 35 

eIF4G2/p97 35, 89 

ELAVL1/HuR 35 

Elk-1 44 

ELP 24 

Emerin 113 

EML4 113 

Endoglin 158 

Endonuclease G 89 

Enolase 142 

eNOS 51, 170 

ENPP1 142 

ENSA 101 

EOMES 158 

EPAC 113 

EpCAM 109, 113 

Eph 69, 170 

EPLIN 113 

Eps8 69 

Eps15 119 

ERC1 174 

ERCC1 101 

EREG 69 

Erk1/2 (p44/42 MAPK) 44 

Erk5 44 

Erlin 113 

Ero1-Lα 119 

ERp44 119 

ERp57 119 

ERp72 119 

ERRα 39 

ESET 24 

Estrogen Receptor 39 

Etk/BMX 69 

ETO 174 

Evi-1 174 

EVL 113 

EWS 24 

Exportin 5 35 

Ezh2 24 

Ezrin/Moesin/Radixin 113 

FAAH1 142 

FABP 142, 187 

FADD 89 

FAF1 89 

FAIM 89 

FAK 109 

FAM129B 44 

Fas 89 

Fas Ligand 89 

Fascin 113 

Fatty Acid Synthase 142 

FE65 187 

FEN-1 101 

Fer 113 

Fes 113 

Fetuin A 142 

Acidic FGF 158, 170 

Basic FGF 158 

FGF Receptor 69, 170 

Fgr 174 

FHIT 142 

Fibrillarin 113 

FIH 158, 170 

Filamin 113 

FIP200 96 

FKBP4 51, 119 

FLCN 51 

FLI1 158 

Flightless-I 158 

FLIP 89 

Flotillin 113 

FLT3 69 

FMRP 35 

α-Fodrin 89 

FosB 44 

c-Fos 44 

Delta FosB 187 

FoxA2/HNF3β 142 

FoxC 142, 158 

FoxD 158 

FoxK 158 

FOXM1 101 

FoxO 51 

FoxP 158, 174 

FRA1 44 

Fragilis 158 

Frizzled 158 

FTH1 142 

Fumarase 142 

FUS/TLS 35 

FXR 35 

Fyn 174 

FYVE-CENT 113 

G9a/EHMT2 24 

Gab 51 

GABA(B)R 77, 187 

GABARAP 96 

GABARAPL2 96 

GAD 187 

GADD45 α 101 

Galectin/LGALS 174 

GAP43 187 

GAPDH 142 

GATA 142, 158 

GCK 45 

GCN2 119 

GCN5L2 24 

GCNF/NR6A1 158 

GEF-H1 113 

Gelsolin 83, 113 

Geminin 101 

GFAP 187 

GFAT 142 

GFI1b 158 

GGA3 187 

GIMAP5 174 

GIT 109, 113 

GKAP 187 

GLDC 142 

GLI 158 

Glucagon 142 

Glucocorticoid Receptor 39 

Glucose-6-Phosphate 142 

Dehydrogenase 

Glut4 142 

Glutamate 

187 

Dehydrogenase 1/2 

Glycogen Synthase 142 

GM130 113, 119 

GNB3 77, 187 

Golgin-97 113 

GOPC 119 

GP130 174 

GPR50 187 

GPX1 142 

GRAF1 187 

Granzyme 89 

Grb2 45 

Grb10 45 

Gremlin 158, 170 

GRK2 187 

GRK6 174 

Grp75 119 

Grp94 119 

GSK-3 51 

GSTP1 187 

GUCY1A2 142 

Gα 77, 83 

GβL 51 

Hamartin/TSC1 52 

HAUSP 123 

HDAC 24 

HECTH9 123 

HEF1/NEDD9 113 

Helios 174 

HELLS 25 

HER/ErbB 69, 70 

Heregulin 158 

hERG1α 142 

HES1 158 

HEXIM1 25 

Hexokinase 142 

HGK 45 

HGS 51 

Hic-5 109 

HIF-1 158, 170 

HIF-2 158 

Hip 119 

HIPK2 89 

HIRA 25 

Histones 24 

HMG (High Mobility 25 

Group) 

HMOX2/HO-2 142 

HNF 142 

hnRNP 35 

HO-1 142, 170 

Homer1 187 

Hop 119 

HP1 25 

HPK1 174 

HR6A/HR6B 101 

HRS 119 

HS1 174 

HSF1 119 

HSL 142 

HSP 119 

HtrA2/Omi 89 

Huntingtin 187 

HYOU1 119 

c-IAP 88 

Cleaved Drosophila 89 

ICE (drICE) (Asp230) 

ID 158 

IDH 142 

IDO 175 

IFI16 174 

IFIT1 174 

IFITM 174 

IFN 174 

IGBP1 174 

IGF-I Receptor 142, 143 

IGF-II Receptor/CI-M6PR 119 

IGFBP 143 

βIG-H3 109 

Ikaros 174 

IκB 174 

IKK 174, 175 

IL (Interleukins) 175 

ILK1 109 

IMP1 35 

Importin β1 113 

INCENP 101 

iNOS 175 

INPP4b 83 

Insulin 143 

Insulin Receptor 143 

Integrin 109, 113, 170 

Interleukins 175 

INTS9 25 

IP3 Receptor 83 

IQGAP 113 

IRAK 175 

IRAP 143 

IRE1α 119 

IRF 175 

IRS 143 

ISG15 123 

ITCH 123 

Itk 175 

IWS1 35 

IκB 174, 175 

Jagged 158, 170 

Jak 175 

JARID 25 

JIP4/SPAG9 45 

JMJD 25 

JNK/SAPK 45 

Jun 45 

K48-linkage Specific 123 

Polyubiquitin 


Polyubiquitin 

KEAP1 123 

Keratin 113 

KHSRP 35 

Ki-67 101 

KIBRA 158 

KIF3A 113, 158 

KIFC1 113 

Kinectin 1 113 

KISS1R 187 

c-Kit 70 

KLF4 158 

KLHL12 123 

KSR1 45 

Ku70 101 

Ku80 101 

La Antigen 35 

Lamin 89, 113 

LAMP1 113 

LAMTOR 51 

Langerin 175 

LAP2a 89 

LASP1 113 

LAT 175 

LAT1 143 

LATS 101 

LC3 96 

Lck 175 

LCMT1 25 

LCP1 113 

LDH-A 143 

LEDGF 25 

LEF1 158 

Lefty1 158 

LGP2 175 

LIMD1 158 

LIMK 113 

LIN28 158 

Lipin 1 143 

5-Lipoxygenase 175 

LIS1 187 

LITAF 175 

Livin 89 


297



LKB1 143 

LLGL1 113 

LMAN1 113 

Lmx1B 158 

LPP 109 

LRF/Pokemon 175 

LRIG1 70 

LRP 158 

LRRK2 187 

LSD1 25 

LSm2 35 

Lsp1 175 

LXR-β 143 

Lyn 175 

Lyric/Metadherin 109 

LysRS 35 

M-CSF Receptor 70 

M-RIP 113 

MacroH2A1 24 

Mad-1 89 

MAD2L1 101 

MAG 187 

Malic Enzyme 143 

MALT1 175 

MAML 158 

Mannose Receptor 175 

MAP2 187 

MAP4K3 51 

MAPBPIP/ROBLD3/p14 35 

MAPKAPK 45 

MAPKSP1/MP1 35 

MARCKS 83 

MARK 113 

Maspin 89, 109, 170 

MASTL 101 

MAVS 175 

Max 89 

MBD3 25 

MBTPS2 119 

MCF2/Dbl 113 

Mcl-1 89 

MCM 101 

MCP-1 175 

MDA-5 175 

MDH2 143 

MDR1/ABCB1 143 

MeCP2 25 

MED 25 

MEF2 45 

MEIS1/2 175 

MEK 45 

MEKK3 45 

MELK 187 

Mena 187 

Menin 25 

MEP50 25 

Mer 70 

MERIT40 101 

Merlin 187 

MESD2 158 

Mesothelin 109 

Met 70 

MetAP2 35 

mGluR1 77, 187 

MGMT 101 

MIB1 123 

Mic-1 158 

Microcephalin-1/BRIT1 101 

β2-microglobulin 174 

Mig6 45 

Mili 35 

Mios 51 

MIS-R2 158 

MITF 158 

Mitofusin 143 

Miwi 35 

Miz-1 175 

MKK 45 

MKP 44 

MLH1 101 

MLK 45 

MLKL 89 

MLLT1/ENL 25 

MMP 109, 170 

MNDA 175 

Mnk1 35 

MO25α/CAB39 143 

MOB1 158 

Moesin 113 

MORF4L1/MRG15 25 

Mre11 101 

MRP/ABCC 143 

MRPL11 35 

MSH 101 

MSK 45 

Mst 89 

Msx1 158 

MTA1 25 

MTAP 143 

MTMR 96 

mTOR 51 

MTSS1 113 

MUC1 109 

Munc18-1 187 

Musashi 187 

c-Myb 158 

Myc 89 

MyD88 175 

Myelin Basic Protein 187 

Myeloperoxidase 175 

MYH 101 

MyoD1 158 

Myosin 113 

Myosin Light Chain 113, 114 

MYPT1 114 

Myt1 101 

Na Channel β1 Subunit 187 

Na,K-ATPase 114 

NAC1 158 

NAE1/APPBP1 123 

Naked 158 

NALP1 175 

Nanog 158 

NBR1 96 

NCAM (CD56) 109 

NCAPD3 101 

NCBP1/CBP80 35 

NCK1 114 

NCoR1 25 

NCS1 187 

NDP52 175 

NDRG 158, 170 

NEDD 123 

NEK7 101 

Nestin 187 

NeuN 187 

NeuroD 187 

Neurofilament 187 

Neurogenin 2 187 

Neuropeptide Y 187 

Neuropilin 170, 187 

NF-κB 175 

NFAT 175 

NFI-C 25 

NG2 187 

NGF 187 

NHERF 187 

Nicastrin 187 

NIK 175 

NIPA 101 

NIPSNAP1 83 

NKCC1 187 

NKX2 158 

NLRC4 175 

NLRP3 175 

NLRX1 175 

NMDAR 187, 188 

NME1/NDKA 143 

Nna1 188 

Nod1 175 

Nogo-A 188 

NOS 175, 188 

Notch 158, 170 

NPC1L1 143 

NPL4 123 

NPM 101 

NQO1 143 

NRBF-2 39 

NRF1 25, 35, 143 

NSF 119 

nSMase1 83 

NT5E/CD73 170, 188 

NTAL/LAB 175 

NTF2 114 

Nucleolin 25 

Nucleomethylin 25 

Nucleostemin 158 

NuMA 101 

Numb 158 

NUP88 114 

NUP98 114 

Nur77 39 

NUT1 25 

NXF1 35 

O-GlcNAc 143 

OCRL1 119 

OGDH 143 

OGT 143 

Oligophrenin-1 188 

Opioid Receptor 77, 188 

ORC 101 

OS-9 119 

OSR1 114 

OTUB1 123 

OTULIN 123 

OTX2 158 

p115 RhoGEF 114 

p14 ARF 101 

p18 INK4C 101 

p190 RhoGAP 114 

p21 Waf1/Cip1 101 

p27 Kip1 101 

p35/25 188 

p38 MAPK 45 

p39 188 

p44/42 MAPK (Erk1/2) 44 

p47phox 175 

p48 Primase 101 

p53 101 

p57 Kip2 101 

p58 Primase 101 

p58IPK 119 

p63-α 101 

p67phox 175 

p70 S6 Kinase 51 

p73 101 

p75NTR 188 

p90RSK1 45 

p95/NBS1 101 

PA28 123 

PABP 35 

PACT 35 

PAF1 25 

Paip2A 35 

PAK 114 

PANK 143 

PAR-4 89 

PAR2 114 

PARK9 188 

Parkin 188 

α-Parvin 109 

PARN 35 

PARP 89 

PASK 143 

PAX 158 

Paxillin 109 

PBK/TOPK 101 

Pbx1 175 

PC1/3 188 

PC2 188 

PCAF 25 

PCK 143 

PCM-1 114 

PCNA 101 

PCTAIRE 1 101 

PD-L1 175 

PDAP1 158 

PDCD4 89 

PDE5 83 

PDGF Receptor 70, 170 

PDHK1 143 

PDI 119 

PDK1 51 

PDLIM2 114 

Pdx1 143 

PEA-15 89 

PELO 101 

PEN2 188 

Perforin 89 

Perilipin 143 

PERK 35, 119 

PFKFB 143 

PFKL 143 

PFKP 143 

PGAM1 143 

PGC1α 143 

PGD 143 

PGRMC1 143 

PHB1 39, 101 

PHC1 25 

PHD-2/Egln1 158, 170 

PHF 25 

PHGDH 143 

PHLDA3 89 

Phospholamban 83 

PI3 Kinase 51, 52 

PI3 Kinase Class II 52 

PI3 Kinase Class III 52 

PI4 Kinase 52 

PIAS 175 

PICH 101 

Pim 175 

Pin1 101 

PINK1 188 

PIP4K2 83 

PIP5K1 83 

Pirin 175 

PiT1/SLC20A1 143 

PITSLRE/CDK11 100 

PKA 83 

PKC 83 

PKD/PKCµ 83 

PKG-1 114 

PKM 143 

PKR 35, 119 

PLA2G1B 143 

PLC 83 

PLD 83 

Plectin-1 114 

Plexin A 188 

PLK 101 

PNK 101 

PNUTS 52 

Podoplanin 114 

POLR3A 25 

Pontin/RUVBL1 25 

PP1α 102 

PP2A 102 

PP2C 45 

PP5 102 

PPARγ 39 

PPIG 35 

PPP1CB 102 

PPP2R 102 

PRAS40 52 

PRC1 114 

Prdx1 143 

Presenilin 188 

PREX1 114 

PRK2 83 

PRMT 25 

Profilin-1 114 

Progesterone Receptor 39 

Prolactin Receptor 175 

Prostate Specific 

119 

Membrane Antigen 

Protor2 52 

PRP4K 35 

PSA/KLK3 109 

PSD93 188 

PSD95 188 

PSMA 123 

PSMB 123 

PSMC 123 

PSMD 123 

PTBP1 35 

PTCH 158 

PTEN 52 

PTK7 70 

PTP1B 143 

PTP4A3 114 

PTPA/PPP2R4 102 

PTPN14 158 

PTPN18 158 

PTPN22 175 

PU.1 175 

Puma 89 

Pumilio 35 

PVR/CD155 114 

Pyk2 45 

Pyruvate Dehydrogenase 143 

Rab 119 

Rabex-5 119 

Rac1/Cdc42 114 

Rac1/Rac2/Rac3 114 

RACK1 114 

Rad 102 

RAD21 25, 102 

Rad23B 123 

Radixin 114 

Raf 45 

RAG1 175 

Rag 52 

RAGE 175 

RAIG1 77, 158 

Ral 114 

RalBP1 114 

Ran 114 

RanBP1 114 

RANK 175 

RANK Ligand 175 

RANTES 175 

Rap1 114 

Raptor 52 

RAR 39 

Ras 45, 114, 158 

Ras-GRF1 188 

RasGRP3 114 

Rb 102 

Rb-like 1 102 

RBAP46 25 

RBBP5 25 

RBPSUH 158 

RBX1 119, 123 

RCAS1 119 

RCC 102, 114 

RCHY1 123 

RECK 109, 170 

RecQ4 102 

RecQL 102 

REDD1 52 

RelB 175 

Renin 109 

REPS1 119 

Reptin/RuvBL2 25 

Ret 70 

Rev-Erba 39 

RGS4 77, 188 

Rheb 52 

Rho 114 

Rhodopsin 188 

Ribosomal Protein 35 

Rictor 52 

Rig-I 175 

RING1 25 

RIP 89, 175 

RKIP 45 

RMP 35 

RNF 25 

ROCK 114 

Ron 70, 170 

ROR 70 

ROS1 70 

RPA 102 

Rpb1 CTD 25, 102 

RPL11 35 

RRM1 102 

RSK 45 

Rubicon 96 

RUNX 158 

RXR 39 

RyR1 83 

S5a/PSMD4 123 

S6 Ribosomal Protein 35 

S100 83 

Sall4 158 

SAM68 35 

SAMHD1 175 

SAP102 188 

Sara 158 

SARM1 175 

SATB1 25 

SAV1 158 

SCAI 114 

SCAP 143 

SCD1 143 

SCF 158 

Scribble 158 

SDF1 175 

SDHA 143 


299



Sec23 114 

Sec24 114 

Sec31 114 

Secretagogin 188 

Securin 102 

SEK1/MKK4 45 

Semaphorin 188 

SENP 123 

Sestrin-2 102 

SET 25 

SF2/ASF 35 

SF3B1 35 

SFRP1 158 

SGK 52 

SGLT1 143 

SGTA 119 

SH2D1A 175 

SHANK2 188 

Sharpin 123 

Shc 45 

Shh/Ihh 158, 159 

SHIP 45, 175 

SHMT 143 

Shootin1 188 

SHP 45, 175 

SIK2 143 

Sin1 52 

SIN3A 25 

SINTBAD 175 

SirT 25 

Siva-1 89 

SIX1 159 

SKAR 35 

Skp 123 

SLBP 102 

SLC1A4 188 

SLC4A4/NBC1 143 

SLC7A11/xCT 143 

SLP76 175 

Slug 159 

Smac/Diablo 89 

Smad 159 

SMARCA 25 

SMARCC 25 

SMC 102 

SMG-1 102 

SMN1 35 

Smurf 159 

SMYD 25 

Snail 159 

SNAP25 188 

SNARK/NUAK2 143 

SNF2H 25 

SNF5 25 

SNIP/p140Cap 119 

SnoN 159 

SOCS 175 

SOD 143, 188 

SOS1 45 

Sox 159 

SP1 25 

SPAK 114 

SPARC 159 

Spartin 102 

SPHK1 83 

SPINK3 123 

Spinophilin 188 

Spry1 70, 170 

SPT 25 

SQSTM1/p62 96 

Src 45 

SRC 25 

SREBP-1c 143 

SRF 45 

SSEA 159 

SSH1 114 

SSRP1 25 

SSTR1 188 

SSU72 25 

STAG2 102 

STAM1 119 

STAMBP 123 

StAR 143 

Stargazin 188 

Stat 175, 176 

Stathmin 114 

STEP 188 

STF-1 39 

STIM 83 

STING 176 

STOP 187 

Succinyl-CoA Synthetase 143 

SUFU 159 

SUMO 123 

Survivin 89 

SUV39H1 25 

SUZ12 25 

Syk 176 

Symplekin 35 

Synapsin 188 

Synaptophysin 188 

Synaptotagmin 188 

SynGAP 188 

Synip 143 

Syntaxin 119, 188 

α-Synuclein 188 

SYVN1 123 

T-bet/TBX21 (V365) 176 

TAB 45 

TACC3 102 

TACE 159 

TAF 25 

TAK1 45 

TAL1 176 

Talin-1 109, 114 

TANK 89 

TAP 176 

Tau 188 

TAX1BP1 89 

TAZ 159 

TBC1D1 143 

TBK1/NAK 176 

TBP 25 

TCEB3/Elongin A 25 

TCF 159 

TCL1 52 

TCTP 102, 114 

TDP43 188 

TEAD 159 

Tec 176 

TECPR1 96 

Tenascin C 170, 188 

Tensin 2 114 

TERF2IP 102 

TESK1 114 

TFAM 188 

TFEB 35 

TFF1/pS2 159 

TFII 25 

Pro-TGF-α 159 

TGF-β 159 

TGF-β Receptor 159, 170 

TGM2 83 

TH1L 25 

Thap11/Ronin 159 

THEMIS 176 

THEX1 35 

Thioredoxin 143 

THOC4/ALY 35 

ThPOK 176 

Thy1 188 

Thymidine Kinase 1 143 

Thymidylate Synthase 143 

Thyroid Transcription 159 

Factor 1 (TTF-1) 

TIAR 35 

Tid-1 119 

Tie2 70, 170 

TIF1β 102 

TIMP1 109, 170 

Tip60 25 

TIRAP 176 

TLE1/2/3/4 159 

TLK1 102 

TMEM49/VMP1 96 

TMP21 188 

TMS1 89 

TNF-α 176 

TNF-R 89, 176 

Tnk1 45 

Toll-like Receptor 176 

Tollip 176 

Topoisomerase IIα 25 

TORC/CRTC 143 

Torsin A 188 

TP/ECGF1 89 

TPH-1 188 

TPOR 176 

TPX2 102 

TRA-1-60 159 

TRA-1-81 159 

TRA-2-54 159 

TRADD 89 

TRAF 89 

TRAIL 89 

Transketolase 143 

TRAP1/HSP75 119 

β-Trcp 123 

TREX1 176 

Trf 102 

TRIAD1 123 

TRIB2 159 

TRIF 176 

TRIM 25, 123, 159 

Trk 188 

Tropomyosin 114 

Troponin 114 

TRPV3 83 

TRRAP 25 

TRXR 143 

TSPO 83 

TTK 102 

TTF1 159 

Tuberin/TSC2 52 

Tubulin 114, 188 

Tug 143 

TWEAK 176 

TWEAK Receptor/Fn14 176 

Twinfilin-1 114 

Tyk2 176 

Tyro3 70 

Tyrosinase 143 

Tyrosine Hydroxylase 188 

U2AF1 35 

UBA2 123 

Ubc 123 

UBE 123 

Ubiquitin 123 


Polyubiquitin 


Polyubiquitin 

Ubiquityl-PCNA (Lys164) 101 

UBLE1A/SAE1 123 

UBR5 123 

UCHL 123 

UGT 143 

UHRF1 25 

ULK1 96 

UNC5B 188 

uPAR 109, 170 

Upf 35 

USP 123 

UTF1 159 

UVRAG 96 

VAMP 119, 188 

VASP 114 

Vav 114 

VCAM1 176 

VCP 102, 123 

VDAC 89 

VEGF 159 

VEGF Receptor 70, 170 

VGLUT 188 

VHL 123, 170 

Villin-1 114 

Vimentin 114 

Vinculin 109 

Vitamin D3 Receptor 39 

VMS1 143 

VPRBP 123 

VRK 102 

Vti1a 188 

WASP 114 

WAVE 114 

Trademarks, Terms and Conditions 

WBP2 159 

WDR5 25 

Wee1 102 

WFS1 83 

WIF1 159 

WIP1 96, 102 

WIPI 96 

WNK 52 

Wnt 159 

WRN 102 

WSTF 25 

WTX 159 

WWOX 89 

XAF1 89 

XBP-1s 35 

XIAP 89 

XLF 102 

XPB 25, 102 

XPC 102 

XPD 25, 102 

XPF 102 

XRCC1 102 

XRN2 35 

YAP 52, 159 

YB1 52 

Yes 176 

YY1 25 

ZAP70 176 

ZFX 159 

ZO 109, 114 

ZPR1 35 

Zyxin 109 

The following are trademarks of Cell Signaling Technology, Inc.: 

AcetylScan, AcetylSignature, eXceptional Monoclonal Technology, eXceptional Performance, Cell Signaling Technology logo, Cell Signaling Technology, CST, 

CST BIOSCIENCES, CST LIFESCIENCES, D4D6, DF53, E1L3N, HTScan, Kinomepath, KinomeView, MethylScan, NG-XMT, PathScan, PhosphoPlus, PhosphoScan, 

PhosphoSignature, PhosphoSite logo, PhosphoSite, PhosphoSitePlus, Phosphotrol, Phototope, PTMScan, SignalFire, SignalKine, SignalSilence, SignalSlide, 

SignalStain, SimpleChIP, StemLight, UbiScan, XMT, XP 

The following are registered trademarks and trademarks are the property of their respective owners: 

Acumen is a registered trademark of TTP Labtech. • Aerius is a registered trademark of LI-COR Biosciences. • Alexa Fluor is a registered trademark of Life 

Technologies Corporation. • AlphaScreen is a registered trademark of PerkinElmer, Inc. • BackDrop is a registered trademark of Molecular Probes, Inc. • Bio-Plex 

is a registered trademark of Bio-Rad Laboratories. • BODIPY is a registered trademark of Molecular Probes, Inc. • DELFIA is a registered trademark of PerkinElmer, 

Inc. • DRAQ5 is a registered trademark of Biostatus Limited. • DRAQ7 is a trademark of Biostatus Limited. • DyLight is a trademark of Thermo Fisher Scientific 

Inc. and its subsidiaries. • EnVision is a trademark of Dako. • ER-Tracker is a trademark of Molecular Probes, Inc. • Gallios is a trademark of Beckman Coulter, Inc. 

• HTRF is a registered trademark of CISbio international. • iBlot is a trademark of Invitrogen. • In-Cell Western is a trademark of LI-COR Biosciences. • LANCE is 

a registered trademark of PerkinElmer, Inc. • LanthaScreen is a trademark of Invitrogen Corporation. • LI-COR is a registered trademark of LI-COR Biosciences. • 

LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories. • Luminex is a registered trademark of Luminex Corporation. • LysoTracker is a registered 

trademark of Molecular Probes, Inc. • MitoTracker is a registered trademark of Life Technologies Corporation. • MULTI-ARRAY is a registered trademark of Meso 

Scale Discovery. • New England Biolabs is a registered trademark of New England Biolabs, Inc. • NovaRED is a trademark of Vector Laboratories. • Odyssey is 

a registered trademark of LI-COR Biosciences. • Pacific Blue is a trademark of Molecular Probes, Inc. • ProLong is a registered trademark of Life Technologies 

Corporation. • Q-View Plus, Q-View, Quansys Biosciences logo are registered trademarks of Quansys Biosciences. • SenseAware is a servicemark of FedEx. • 

Sepharose is a registered trademark of GE Healthcare. • SNAPi.d. is a trademark of Millipore. • Stratalinker is a registered trademark of Stratagene. • SYBR Green 

is a registered trademark of Molecular Probes, Inc. • Triton is a trademark of The Dow Chemical Company or an affiliated company of Dow. • Trizma is a registered 

trademark of Sigma-Aldrich Co. LLC. • Tween is a registered trademark of ICI Americas, Inc. • UniProt is a trademark of European Molecular Biology Laboratory. 

Please visit www.cellsignal.com/tm for a current listing of applicable trademark information. 

Use of CST Motif Antibodies within certain methods (e.g., U.S. Patent No.’s 7,198,896 and 7,300,753) may require a license from CST. For information regarding 

academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial 

licensing terms please contact CST Pharma Services at marketing@cellsignal.com. 

The use of reagents from Cell Signaling Technology in conjunction with the technologies identified throughout this guide may require a license to third party 

intellectual property rights. 

Alexa Fluor dye antibody conjugates are sold under license from Life Technologies Corporation, for research use only for immunocytometry, immunohistochemistry, 

high content screening (HCS) analysis, or flow cytometry applications, and excluding use in combination with DNA microarrays and high content screening (HCS). 


301

32 


Diagram & Table Keys 

and additional resources 

Applications Key 

While all of our antibodies are rigorously tested in a number of relevant applications, some products are more suitable 

for a specific application. This information is summarized in various lists and tables found throughout this guide. 

chapter 32: Diagram & Table Keys 

Pathway Diagram Key 

The pathway diagrams found in this guide and on our website have been assembled by CST scientists and outside 

experts to provide succinct and current overviews of selected signaling pathways. 

Direct Stimulatory Modification 

Direct Inhibitory Modification 

Deacetylase 

Ribosomal subunit 

WB Western Blotting 

IP Immunoprecipitation 

IHC Immunohistochemistry 

IF Immunofluorescence 

F Flow Cytometry 

ChIP Chromatin Immunoprecipitation 

-IC Immunocytochemistry 

-P Paraffin 

-F Frozen 

E-P Peptide ELISA 

Multistep Stimulatory Modification 

Multistep Inhibitory Modification 

Tentative Stimulatory Modification 

Tentative Inhibitory Modification 

Separation of Subunits 

or Cleavage Products 

Joining of Subunits 

Translocation 

Transcriptional Stimulatory Modification 

Transcriptional Inhibitory Modification 

Kinase 

Phosphatase 

Transcription Factor 

Caspase 

Receptor 

Enzyme 

pro-apoptotic 

pro-survival 

GAP/GEF 

GTPase 

G-protein 

Acetylase 

TIM-3 

Galectin-9 

B7-H3 

B7-H4 

CTLA-4 

CD80, 86 

PD-1 

PD-L1 

TCR 

MHC 

ICOS 

ICOSL 

OX40 

OX40L 

CD40 

CD40L 

CD27 

CD70 

CD137 

CD137L 

CD28 

Reactivity Key 

H human 

M mouse 

R rat 

Hm hamster 

Mk monkey 

C chicken 

Mi mink 

Dm D. melanogaster 

X Xenopus 

Z zebra fish 

Amino Acid Properties Table 

B bovine 

Dg dog 

Pg pig 

Sc S. cerevisiae 

Name 

Single 

Abbreviation letter code MW pKa Codons 

Side chain 

characteristics Modifications 

Glycine Gly G 57.05 na G-G-(UCAG) Nonpolar 

Alanine Ala A 71.09 na G-C-(UCAG) Nonpolar 

Valine Val V 99.14 na G-U-(UCAG) Nonpolar 

Leucine Leu L 113.16 na C-U-(UCAG), U-U-(AG) Nonpolar 

Isoleucine Ile I 113.16 na A-U-(UCA) Nonpolar 

Phenylalanine Phe F 147.18 U-U-(UC) Nonpolar 

Proline Pro P 97.12 na C-C-(UCAG) Nonpolar Racemization 

Serine Ser S 87.08 16 A-G-(UC), U-C-(UCAG) Hydroxyl group Phospho-, Glycosyl-, 

Eliminyl- 

Threonine Thr T 101.11 7.6 A-C-(UCAG) Hydroxyl group Phospho-, Glycosyl-, 

Eliminyl- 

Tyrosine Tyr Y 163.18 na U-A-(UC) Hydroxyl group Adenyl-, Phospho-, 

Sulfo- 

Cysteine Cys C 103.15 8.35 U-G-(UC) Contains sulfur Eliminyl- 

Methionine Met M 131.19 na A-U-G Contains sulfur 

Asparagine Asn N 114.11 na A-A-(UC) Contains nonbasic 

nitrogen 

Glutamine Gln Q 128.14 na C-A-(AG) Contains nonbasic 

nitrogen 

Tryptophan Trp W 186.21 na U-G-G Contains nonbasic 

nitrogen 

Aspartic Acid Asp D 115.09 3.9 G-A-(UC) Acidic 

Glutamic Acid Glu E 129.12 4.07 G-A-(AG) Acidic 

All all species 

expected 

( ) 100% sequence 

homology 

Glycosyl-, Deamidation 

Deamidation 

Lysine Lys K 128.17 10.79 A-A-(AG) Basic Acetyl-, Adenyl-, 

Carbamyl-, Methyl- 

Ubiquitin, SUMO, ISG, 

Neddy, UFM1 

Arginine Arg R 156.19 12.48 A-G-(AG), C-G-(UCAG) Basic Methyl-, Citrulline 

Histidine His H 137.14 8.04 C-A-(UC) Basic Adenyl-, Phospho- 


303

33 


Contact Information 

Global Headquarters 

3 Trask Lane, Danvers, MA 01923 USA 

Phone: 978-867-2300 | Fax: 978-867-2400 | www.cellsignal.com 

Stay Informed! 

Request monthly updates on the latest CST products and scientific resources. www.cellsignal.com/subscribe 

Ordering and Customer Service 

Phone: 877-616-2355 [US & Canada toll free] or 978-867-2388 

Email: orders@cellsignal.com | Fax: 978-867-2488 | Order Online: www.cellsignal.com 

Find Your Local Distributor: www.cellsignl.com/contactus 

Technical Support 

At CST, the same Product Scientists who produce and validate our products are available to help you with your 

research needs, openly sharing their expertise and data. 

Online support request form: www.cellsignal.com/support 

Phone: 877-678-8324 [toll-free in US, Canada] or 978-867-2388 | Fax: 978-867-2400 

Request a custom formulation visit: www.cellsignal.com/custom 

Product scientists are available 9:00 am – 6:00 pm (EST), Monday–Friday 


305

“…the time has come for us... to puzzle out, 

one protein at a time, how signals are really 

processed inside cells to create the marvelously 

functioning apparatus – the eukaryotic cell.” 


Daniel K. Ludwig Professor for Cancer Research at MIT 

GDE2015ENG_00 

www.cellsignal.com 

Printed in the USA on 100% recycled paper (30% post-consumer) waste fiber. Cover Image: Vesicle Trafficking Cellular Landscape, see pg for more information 248.

CST Guide:

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