Title Page
Contents
CHAPTER-1. [제목없음] 6
Contents 6
국문초록 11
I. Introduction 12
II. Materials and Methods 14
A. Cell Culture and Transfection 14
B. Immuno fluorescence microscopy 14
C. Immunoblot Analysis 14
D. Antibodies 15
E. Knock down 15
F. Polysome Analysis 15
G. Co-Immunoprecipitation Assay 16
H. Proximity Ligation Assay (PLA Assay) 16
III. Results 17
A. Ca2+/Calmodulin signaling involvement in Stress granule assembly[이미지참조] 17
B. P-MAPK assay Screening 17
C. Calmodulin and AKT are components of stress granules 19
D. Calcium regulates AKT activation 19
E. Ca2+/ Calmodulin complex is needed for eIF2α phosphorylation[이미지참조] 23
F. AKT is an interaction partner of CaM and eIF2α 23
IV. Discussion 31
REFERENCES 40
ABSTRACT 45
CHAPTER-2. Analysis of MeCP2 protein in Stress Granule Assembly 46
Contents 47
국문초록 50
I. Introduction 51
II. MATERIALS AND METHODS 53
A. Cell Culture and Transfections 53
B. Immuno fluorescence microscopy 53
C. Immunoblot Analysis 53
D. Knock down 54
E. Cell fractionation protocol 54
F. Antibodies 54
G. Polysome Analysis 54
H. Immunoprecipitation Assay 55
I. IDR/IDP Analysis with IUPred2A tool 55
J. Statistical analysis 55
III. Results and Discussion 56
A. MeCP2 is a component of Stress granules 56
B. Subcellular fractionation to see the existence of MeCP2 in Cytosol 56
C. Intrinsically disordered protein (IDP) profile of MeCP2 61
D. Association of MeCP2 and its domains in polysome distribution 63
E. MeCP2 and its domains exhibit both impaired SG nucleation and recruitment to SGs in U2OS cells 63
IV. REFERENCE 71
V. ABSTRACT IN ENGLISH 76
CHAPTER-1 10
Fig. 1. Ca2+/Calmodulin signaling involvement towards stress granule assembly.[이미지참조] 18
Fig. 2. Calmodulin and AKT are components of SG's. 20
Fig. 3. Arsenite induced polysome disassembly is recovered due to CaM antagonist W5... 21
Fig. 4. Overexpression of Calmodulin doesn't affect polysome disassembly either in non-... 22
Fig. 5. Calmodulin is required for AKT activation and eIF2α phosphorylation. 24
Fig. 6. Calcium is required for AKT activation and eIF2α phosphorylation. 25
Fig. 7. AKT knock down impairs stress granule assembly. 26
Fig. 8. AKT interacts with Calmodulin and eIF2α upon arsenite treatment. 27
Fig. 9. AKT is necessary for eIF2α/ATF4 signaling pathway. 30
CHAPTER-2 49
Fig. 1. MeCP2 proteins are components of stress granules. 57
Fig. 2. MeCP2 proteins knock down affect stress granule assembly. 58
Fig. 3. MeCP2 exist in cytoplasmic extracts of different cell lines. 59
Fig. 4. Polysome Analysis with SH-SY5Y cells. 60
Fig. 5. IDR prediction profile of MeCP2 62
Fig. 6. Knock out of MeCP2 doesn't show any changes in polysome disassembly. 64
Fig. 7. Stress granule inhibition was not seen in HAP1-MeCP2-KO cell line on Arsenite condition. 65
Fig. 8. Polysome association of MeCP2 and its domains were analysed 66
Fig. 9. Polysome association of MeCP2 and its domains were analyzed 67
Fig. 10. MeCP2 and its domains exhibit both impaired SG nucleation and recruitment to... 69
Fig. 11. Schematic representation of MeCP2 domain constructs 69
Fig. 12. MeCP2 Co-localize with SG marker G3BP in SH-SY5Y cells. 70
Fig. 1. Calcium chelator EGTA inhibits stress granule assembly. 33
Fig. 2. Calcium chelator BAPTA-AM inhibits stress granule assembly. 34
Fig. 3. Blocking of Calcium channels by LaCl3 inhibits SG formation upon Arsenite Treatment. 35
Fig. 4. Arsenite induced Stress granules was inhibited by Ruthenium Red (RuR) treatment. 37
Fig. 5. The Human Phospho-MAPK Array shows the effect of Calmodulin inhibitors on... 38
Fig. 6. The Human Phospho-MAPK Array shows the knock down efficiency of Calmodulin... 39