LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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Figure 4.3.15: 14-3-3γ loss does not alter the localization of adherens junction, gap junction and polarity proteins at the cell border. (A) HCT116 derived vector control or 14-3-3γ knockdown clones were fixed and immuno stained with the indicated antibodies (E-Cadherin, P-Cadherin, ZO-1, Par3 and β-catenin). Please note that cell border recruitment of any of the adherens junction proteins are not altered in 14-3-3γ knockdown clones as compared to vector control cells. (B)The mean fluorescence intensities at the cell borders were measured for 24 cells in 3 independent experiments for each immunofluorescence analysis. (Original magnification x 630 with 2X optical zoom). 134
Figure 4.3.16: Localization of PKP3 at the cell border is altered in 14-3-3γ knockdown testes. Testes sections from mice injected with viruses encoding the vector control or a shRNA targeting 14-3-3γ were stained with antibodies to PKP3. Note that the cell border staining of PKP3 is reduced in the testis of both 14-3-3γ knockdown mice as compared to vector control. 135
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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%
Page 83 and 84: mounting agent. Confocal images wer
Page 85 and 86: 3.26 Antibodies used for Western bl
Page 87 and 88: each plate was added cold 500ml of
Page 89 and 90: 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: Figure 4.3.13: 14-3-3γ loss leads
Page 137 and 138: Figure 4.3.18: Reintroduction of sh
Page 139 and 140: A B 139
Page 141 and 142: from our laboratory demonstrated th
Page 143 and 144: Figure 4.3.21: Plakoglobin recruitm
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
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transgenic research and the use of
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(reviewed in (152, 374)). Kinesins
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subtypes known to date. Type IV col
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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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