臺灣博碩士論文加值系統

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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