Title Page
Contents
Nomenclature 11
General Abstract 13
General Introduction 15
CHAPTER Ⅰ. Phosphorylation of rpS3 by Lyn increases translation of Multi-Drug Resistance (MDR1) gene 28
Abstract 29
Abstract in Korean 30
Introduction 31
Materials and Methods 36
1. Antibodies and reagents 36
2. Plasmids 37
3. Site-directed mutagenesis 39
4. Protein expression and purification 40
5. Cell culture and Transfections 41
6. Cell culture of doxorubicin-resistant MPC11 cell line (MPC11-DOX) 41
7. Ribosome Fractionation 42
8. Reverse Transcription Polymerase Chain Reaction (RT-PCR) 42
9. In vitro binding assay 43
10. Immunoblotting 44
11. Immunoprecipitation 45
12. In vitro Kinase assay 46
13. Drugs and preparation of pervanadate 46
14. Protein synthesis analysis 47
15. Statistical analysis 48
Results 49
Discussion 76
CHAPTER Ⅱ. Production, Characterization, and Epitope Mapping of Monoclonal Antibodies of Ribosomal Protein S3 (rpS3) 80
Abstract 81
Abstract in Korean 82
Introduction 83
Materials and Methods 87
1. Overexpression and purification of rpS3 recombinant proteins 87
2. Production of monoclonal antibodies 93
3. Peptide synthesis 93
4. Western blotting 96
5. Cell culture and siRNA transfection 97
6. Direct ELISA assay 97
7. Direct ELISA of synthesized peptides 98
8. Sandwich ELISA 99
9. Competitive ELISA. 100
10. Immunoprecipitation 100
Results 102
DISCUSSION 125
References 128
List of Publications 134
CHAPTER Ⅰ 38
Table 1. PCR primers used in the PCR in this study 38
Table 2. Schematic diagram of rpS3 mutations used in this study. 40
Table 3. PCR primers used in real time PCR 43
CHAPTER Ⅱ 89
Table 4. Primer sequences for PCR in this study. 89
Table 5. Primer list of rpS3(211-238) recombinant protein fragment. 91
Table 6. Peptide synthesis for peptide scanning. 94
General Introduction 16
Figure 1. The primary structure of the ribosomal protein S3. 16
Figure 2. Regulation of Lyn Localization and Activation in Lipid Rafts and SFK Inhibition by CD45. 20
Figure 3. Advances in understanding the role of P-gp in doxorubicin resistance. 22
Figure 4. Predictive Antibody and Epitope Design through Shared Structure-Based Motifs at the Paratope-Epitope Interface. 25
CHAPTER Ⅰ 51
Figure 5. RpS3 interacts with Lyn in vitro. 51
Figure 6. Identification of interaction domain of rpS3 with Lyn. 54
Figure 7. Ribosomal protein S3 (rpS3) interacts with Src homology 3 (SH3) domain of Lyn. 56
Figure 8. Identification of interaction domain of rpS3 with Lyn. 59
Figure 9. Lyn interacts with C-terminal end of rpS3. 60
Figure 10. RpS3 phosphorylates Lyn at Y167 residue. 64
Figure 11. Binding activity of rpS3 to Lyn is increased by the addition of doxorubicin. 65
Figure 12. Tyrosine phosphorylation of rpS3 is increased by the addition of Ara-C and is decreased by the inhibitors of tyrosine kinase. 68
Figure 13. Characterization of MPC11-DOX (doxorubicin 60 nM) resistant cells. 71
Figure 14. Identification of activity translated MDR1 mRNA. 74
Figure 15. Schematic model describing the increased p-glycoprotein synthesis by increased translation of multi-drug resistance gene (MDR1)... 79
CHAPTER Ⅱ 103
Figure 16. The amino acid sequence and a diagram of rpS3. 103
Figure 17. Identification of epitope binding region using monoclonal antibodies against rpS3. 106
Figure 18. The conformational epitope of pAb R2 was identified using competitive ELISA. 110
Figure 19. Epitope mapping of mAb M7 and pAb R2 in synthesized peptides. 112
Figure 20. Hybridoma clones for monoclonal antibody production were identified using Western blotting and direct ELISA. 116
Figure 21. Epitope mapping of mAb M7 and pAb R2 in the recombinant proteins His-S3 (FL) and GST-S3-P (C-terminal region) was performed using direct ELISA and sandwich ELISA. 120
Figure 22. Identification of immunoprecipitation of mAbs in various proteins. 123