LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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whole cell extract (WCE) shows one tenth input. (B) HCT116 cells were transfected with HA tagged 14-3-3γ or the vector (pCDNA3). Protein extracts were prepared 48 hours post transfection and immunoprecipitations (IP) performed with antibodies to the HA epitope. The total extracts (WCE) and IP’s were resolved on SDS-PAGE gels followed by Western blotting with antibodies to HA and KIF5B. 4.3.11 PG recruitment to cell border is microtubule dependent. As 14-3-3γ can interact with both plakoglobin and KIF5B we hypothesized that PG is recruited to the cell border byKIF5B in a microtubule dependent manner and 14-3-3γ is required to load PG onto KIF5B. To test this hypothesis we first asked whether PG recruitment to cell border is microtubule dependent both in the presence or absence of calcium as previous results from the laboratory have demonstrated that PG is present at cell border in HCT116 cells both in presence or absence of calcium. To test this hypothesis, HCT116 cells were cultured in low calcium medium in presence or absence of nocodazole which induces microtubule depolymerization for 2 hours. An immunoflouresence analysis was performed using specific antibodies to PG and α- tubulin. As shown in figure 4.3.21 (A and B) the cell border recruitment of PG is compromised in the cells treated with nocodazole and the decrease in the cell border recruitment of PG is statistically significant (Figure 4.3.21 B). The nocodazole used in the experiment was sufficient to depolymerize microtubules as can be seen by the absence of microtubules (Figure 4.3.21 A and C). These results suggest that PG recruitment to the cell border is microtubule dependent. We also stained the HCT116 derived vector control 14-3-3γ knockdown cells with antibodies to KIF5B and α-tubulin. We found that there is no difference in either the expression or localization of KIF5B or α-tubulin in HCT116 derived vector control and 14-3-3γ knockdown clones (Figure 4.3.22). We found that in both vector control and 14-3-3γ knockdown clones KIF5B decorates the α-tubulin filaments. 142
Figure 4.3.21: Plakoglobin recruitment to the cell border is microtubule dependent. (A) HCT116 cells were grown in low calcium medium in presence or absence of nocodazole (16µM) for 2 hours. The immunoflouresence analysis was performed using specific antibodies to PG and α-Tubulin. Note that localization of plakoglobin is altered in the cells treated with Nocodazolenocodazole. α-tubulin staining shows that the microtubules are depolymerized upon treatment with Nocodazolenocodazole.(B) The mean fluorescence intensities at the cell borders for PG were measured for 24 cells in 3 independent experiments for each immunofluorescence analysis.(C) The total fluorescence intensities for α-tubulin were measured for 24 cells in 3 independent experiments for each immunofluorescence analysis. . (Original magnification x 630 with 2X optical zoom). 143
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Generation of knockdown mice that l
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STATEMENT BY AUTHOR This dissertati
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CERTIFICATE I certify that the thes
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I am thankful to Mr Uday Dandekar f
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3.16 Isolation of Genomic DNA (gDNA
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A synopsis of thesis to be submitte
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esults in loss of 14-3-3 binding an
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and NheI (NEB) to remove tetO-CMV,
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Hanging drop and wound healing assa
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360). To generate transgenic animal
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asement of the seminiferous tubules
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normal female, pups were screened f
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14-3-3ε only in the presence of HP
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proteins are expressed in testis (3
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5. Lalit Sehgal, Srikanth B., Khyat
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Presented a poster at 33rd All Indi
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shRNA siRNA TBS-T U.V WT C IF DSG D
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List of Figures: Page No 1. Figure
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37. Figure 4.3.20: 14-3-3γ interac
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CHAPTER 1 INTRODUCTION 39
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1.1.1 Cyclin Cdk complexes Eukaryot
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1.1.1.c Cyclin A cdk complexes The
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of which are associated with acquis
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1.2.2 The G1/S DNA Damage checkpoin
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complexes. DNA damage induced degra
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cytoplasmic accumulation of cdc25 p
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14-3-3 binding site. 14-3-3 binding
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G2 arrest and induced premature chr
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emoval of S216 phosphorylation as i
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embryogenesis, a loss of cdc25A can
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dimer was essential for Raf activat
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of enhanced apoptosis of peripheral
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the generation of transgenic mice a
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CHAPTER 2 AIMS AND OBJECTIVES 67
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CHAPTER 3 MATERIALS AND METHODS 69
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Name of oligonucleotide EF1α Forwa
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cleavage sites GAAGGTATATTGCTGTTGAC
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R E1 A GAACGAATAGTGAAGCCACAGATGTA s
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100mm 25ug 225ul 250ul 500 1000ul T
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for the transgene were considered a
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solution in TBS-T containing 0.02%
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mounting agent. Confocal images wer
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3.26 Antibodies used for Western bl
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each plate was added cold 500ml of
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3.32 Hanging drop assay to determin
Page 91 and 92: 3.37 Biodistribution of 18 F -FDG u
Page 93 and 94: 2.5M NaCl 150mM 60ml Tween-20 (Sigm
Page 95 and 96: CHAPTER 4 RESULTS 95
Page 97 and 98: Figure 4.1.1 Generation of lentivir
Page 99 and 100: 4.1.2 Generation of lentiviral vect
Page 101 and 102: transduction with the virus. Untran
Page 103 and 104: Figure 4.1.6: Application of bi-cis
Page 105 and 106: Figure 4.1.7: Application of bi-cis
Page 107 and 108: Figure 4.2.1: Infection of morulae
Page 109 and 110: Figure 4.2.2: Generation of transge
Page 111 and 112: Figure 4.2.3: EGFP-f expression in
Page 113 and 114: sequenced and the distribution amon
Page 115 and 116: 4.3 Generation of 14-3-3γ knockdow
Page 117 and 118: numbering). Alignment was done usin
Page 119 and 120: presence of EGFP-f by performing po
Page 121 and 122: the mouse tested infected with vect
Page 123 and 124: Figure 4.3.7: Staging of 14-3-3γ k
Page 125 and 126: containing spermatozoa is on the Y-
Page 127 and 128: Figure 4.3.9: 14-3-3γ knockdown le
Page 129 and 130: Figure 4.3.11: 14-3-3γ knockdown i
Page 131 and 132: intercellular space (12, 110, 115,
Page 133 and 134: Figure 4.3.13: 14-3-3γ loss leads
Page 135 and 136: Figure 4.3.16: Localization of PKP3
Page 137 and 138: Figure 4.3.18: Reintroduction of sh
Page 139 and 140: A B 139
Page 141: from our laboratory demonstrated th
Page 145 and 146: 4.3.23 (A and B)). These proteins g
Page 147 and 148: the desmosomal proteins recruitment
Page 149 and 150: Figure 4.3.25: Generation of stable
Page 151 and 152: KIF5B, however its reduction does n
Page 153 and 154: compared to vector control cells. (
Page 155 and 156: 4.4 Generation of 14-3-3ε knockdow
Page 157 and 158: into the interstitial spaces of the
Page 159 and 160: Figure 4.4.3: 14-3-3ε knockdown in
Page 161 and 162: Figure 4.4.4: Patchy hair loss in 1
Page 163 and 164: follicle formation marked by black
Page 165 and 166: Figure 4.4.7: Phenotypes in 14-3-3
Page 167 and 168: compared to WT lungs (indicated by
Page 169 and 170: compared to wild type (Figure 4.4.1
Page 171 and 172: and CD4 positive lyumphocyte number
Page 173 and 174: Figure 4.4.13: Bone marrow cells fr
Page 175 and 176: Figure 4.4.15: Generation of stable
Page 177 and 178: Figure 4.4.16: NIH3T3 cells lacking
Page 179 and 180: Figure 4.4.18: Generation of induci
Page 181 and 182: CHAPTER 5 DISCUSSION 181
Page 183 and 184: to generate transgenic animals expr
Page 185 and 186: transgenic research and the use of
Page 187 and 188: (reviewed in (152, 374)). Kinesins
Page 189 and 190: subtypes known to date. Type IV col
Page 191 and 192: Figure 5.1 Schematic representation
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loss of 14-3-3ε the T cell activat
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BIBLIOGRAPHY: 1. Abraham, R. T. 200
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29. Blomer, U., L. Naldini, T. Kafr
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58. Cheng, C. Y., and D. D. Mruk. 2
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90. Elia, A. E., P. Rellos, L. F. H
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118. Girard, F., U. Strausfeld, J.
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150. Herrmann, H., M. Haner, M. Bre
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and activation of a cyclin E-cdk2 c
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213. Liu, D., J. Bienkowska, C. Pet
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242. Moll, R., W. W. Franke, D. L.
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274. Pagano, M., R. Pepperkok, F. V
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305. Russell, L. D. 1978. The blood
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inhibitor of metalloproteases-1 in
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364. Tsunekawa, N., M. Matsumoto, S
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397. Yashiro, M., N. Nishioka, and
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Research: Biology to Prevention to
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LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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