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I. Introduction 17
II. Literature review 33
III. Materials and Methods 39
1. Cell line 39
2. Antibodies 39
(1) Fe65 39
(2) Tau 40
3. DNA constructs and site-directed mutagenesis 40
(1) Fe65 40
(2) Tau 41
4. Transfection and immunoprecipitation 41
(1) Fe65 41
(2) Tau 42
5. Phoshporylation of recombinant Fe65 43
6. Co-immunoprecipitation of SUMO with Tau 43
7. Non-Radioactive Protein Kinase Assay of SGK1 with FITC-Fe65 peptide 45
8. Double Immunofluorescence Mcroscope. 45
(1) Fe65 45
(2) Tau 46
9. In vitro SUMO-1 conjugation assay. 47
10. Generation of anti-phospho-Ser566 Fe65.(이미지참조) 48
11. SUMO and Tau amino acid sequence comparison analysis. 49
IV. Results 51
1. Fe65 51
(1) Phosphorylation of the recombinant Fe65 proteins and the Fe65 peptides by SGK1 51
(2) The Phospho 566Ser Fe65-specific antibody(이미지참조) 54
(3) Comparison of subcellular localization of wild type Fe65 and Fe65 and Fe65S566A mutant(이미지참조) 56
(4) Cofocal microscopy with the phospho-specific antibody against the phosphorylated 566 Ser Fe65(이미지참조) 58
2. Tau 63
(1) The Sub cellular localization of 214-serine phosphorylated Tau in COS-1 Cell 63
(2) Tau SUMOylation in vitro 68
(3) Confocal microscopic analysis of Tau SUMOylation 71
(4) Confocal microscopic analysis of Tau ubiquitinization 74
(5) Tau SUMOylation in COS-1 cell 77
(6) Tau protein stability 81
(7) Cell viability change 83
V. Discussion 85
1. Phosphorylation of Fe65 and its physiological implication 85
2. Phosphorylation of Tau and its physiological implication 88
VI. Conclusion 95
1. Fe65 95
2. Tau 96
VII. References 99
감사의 글 113
Table 1. FACS results with Tau mutants. 84
Fig 1. Functional domains of Fe65 in the rat brain. In this study, a Fe65 variant (AY30550 ; NCBI Accession Number) was used 18
Fig 2. Sequence homology of Tau like protein in plants. (A) Multiple alignments of Tau with Tau like protein in Arabidopsis thaliana and Oryza sativa at the level of amino acid. 23
Fig 3. Sequence homology of SUMO like protein in plant. (A) Multiple alignments of SUMO with SUMO like protein in Arabidopsis thaliana at the level of amino acid.... 29
Fig 4. Organized DataBase method of Plant amino acid and analysis method of genetic information. To find homology of plant and SUMO, Tau sequence, these related amino acid sequences of plant... 49
Fig 5. The phosphorylation of Fe65 by SGK1. (A) One hundred ng of the purified Fe65 PTB2 fragment (39 kDa) or Fe65 PTB2 Ser566 were incubated with one hundred ng of active SGK1(Upstate...(이미지참조) 53
Fig 6. The immunoblotting with the phosphorylated 566Ser Fe65-specific antibody. COS-1 cells were transiently transfected with expression vectors containing cDNA for myc...(이미지참조) 55
Fig 7. The subcellular localization of wild type Fe65 (A) and Fe65S566A mutant (B). COS-1 cells were transiently transfected with expression vectors containing cDNA for myc...(이미지참조) 57
Fig 8. Confocal microscopy with the phosphorylated 566Ser Fe65 specific antibody. COS-1 cells were transiently transfected with expression vectors containing the cDNA for myc epitope-tagged...(이미지참조) 61
Fig 9. Summary of Tau domains and mutant used in SUMOylation study. Two major domains (Projection and association domain) are in Human Tau protein 40... 64
Fig 10. The nuclear or nuclear vicinity localization of 214 Ser phosphor Tau. Tau, a microtubule associated protein, was detected along with microtubule fiber (A, left).... 66
Fig 11. Tau 340K residue in 339VKSE342 is required for its multiple SUMOylation in vitro. The SUMOylation site (340K in 339VKSE342) was noticed in its tubulin binding domains, as...(이미지참조) 70
Fig 12. The observation of Tau wt, S214E, S214A or Tau S214E K340R SUMOylation with the confocal microscopic analysis.... 73
Fig 13. The Confocal microscopic analysis of Tau wt, S214A, S214E or Tau S214E K340R ubiquitinization. The confocal microscopic analysis of transfected EGFP-Tau wt, S214A,... 76
Fig 14. Tau S214E an analog of the phosphorylated Tau at S214, promotes Tau SUMOylation in vivo. To confirm again Fig. 10 observation which Tau S214E is more effectively... 79
Fig 15. The comparison of Tau protein stability with pulse-chase experiments. Tau and Tau-S214E were transfected into COS-1 cells and cells were treated with cyclohexamide.... 82
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