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研究生:鄭侑玲
研究生(外文):Yu-LingCheng
論文名稱:以影像呈現探究細胞中磷酸水解酶PP2A催化次單元及與其有交互作用的蛋白分子所形成的複合體
論文名稱(外文):Visualizing the Complexes Formed by Protein Phosphatase 2A Catalytic Subunit (PP2Ac) and its interacting protein in cells
指導教授:蔣輯武
指導教授(外文):Chi-Wu Chiang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:82
中文關鍵詞:蛋白質磷酸酶雙分子螢光互補法能量共振轉移法
外文關鍵詞:PP2AMID1Alpha4TIPBiFCFRET
相關次數:
  • 被引用被引用:0
  • 點閱點閱:179
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  • 下載下載:0
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中文摘要
Abstract
List of contents .........................................................................................................................I
List of Figures .......................................................................................................................III
List of Tables ........................................................................................................................ V
List of Abbreviations......................................VI
Introduction .........................................................................................................................1
Reverse phosphorylation regulates cellular activities..................................................2
Protein phosphatase 2A (PP2A) .........................................................................................................................2
The canonical PP2A holoenzyme .........................................................................................................................2
The non-canonical PP2A subunit: Alpha4 (α4) ............................................................3
The ubiquitin E3 ligase:
MID1.....................................................................................................................4
The non-canonical PP2A subunit: TIP......................................................................................................................6
Bimolecular Fluorescence Complementation (BiFC) and Fluorescence Resonance Energy Transfer (FRET)...................................................................................................................6
Materials and Methods .........................................................................................................................9
Antibodies and sepharoses ........................................................................................................................10
Materials...............................................................................................................11
Western Blotting ........................................................................................................................11
Cell culture ........................................................................................................................13
Immunefluorescence......................................................................................................14
Inhibitors ........................................................................................................................14
Methods.................................................................................................................15
Cell culture.................................................................................................................15
Transfection by Lipofectamine 2000 (Invitrogen) ...........................................................15
Immunofluorescence (IF) ........................................................................................................................16
Immunopreicipitation (IP) ........................................................................................................................17
Establishing BiFC expression constructs and performing BiFC analysis...................................................17
Establishing Cyan Fluorescence Protein (CFP)-fused expression
constructs and performing BiFC-based FRET analysis ...........................................................18
Results ........................................................................................................................25
Investigating subcellular localization of PP2Ac and its binding proteins...........................................26
Investigating the interaction between α4, MID1 and PP2Ac by BiFC analysis..............................................28
Investigating the interaction between MID1 and TIP........................................................29
Visualizing association and localization of the ternary PP2Ac/α4/MID1
complex by BiFC based-FRET analysis ...........................................................30
Conclusion ........................................................................................................................31
Discussion ........................................................................................................................32
Visualization of the subcellular localization of non-canonical PP2A complexes
by BiFC and BiFC-based FRET assay ...........................................................33
Alpha4 protein, a scaffold of PP2Ac to interact with MID1.......................................................34
MID1/TIP, a novel complex? ...........................................................35
References ........................................................................................................................37
Figures ........................................................................................................................42
Tables ........................................................................................................................62
Appendix ........................................................................................................................66

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