Title Page

Contents

ABSTRACT (in English) 10

1. INTRODUCTION 12

2. MATERIALS AND METHODS 15

2.1. Materials 15

2.2. Cell culture 16

2.3. Western blot analysis 17

2.4. Immunoprecipitation (IP) 17

2.5. ROS assay 18

2.6. Preparation of cytosolic and nuclear fractions 18

2.7. Small interference siRNA transfection 19

2.8. Site directed mutagenesis 19

2.9. Chromatin immunoprecipitation (ChIP) and ChIP-PCR 20

2.10. Confocal microscopy 21

2.11. Cell migration assay 21

2.12. Purification of recombinant protein 22

2.13. In vitro kinase assay 22

2.14. Cells Proliferation Measurement by MTT Reagents 23

2.15. Statistical analysis 23

3. RESULTS 24

3.1. LPS induces superoxide production through RhoA/ROCK, leading to NF-κB activation 24

3.2. Phosphorylated p65/RelA at Ser536 residue binds with p-Tyr42 RhoA. 28

3.3. p-Ser536 p65/RelA and p-Tyr42 RhoA translocate to nucleus in response to LPS stimulation. 30

3.4. LPS induces PGK1 through p-Tyr42 RhoA and p-65. 32

3.5. p-Ser536 p65/RelA and p-Tyr42 RhoA bind to promoter of PGK1 gene. 35

3.6. PGK1 leads to β-catenin phosphorylation during inflammation process upon LPS. 37

4. DISCUSSION 42

5. CONCLUSION 46

References 47

APPENDICES 50

Appendix A. Supplementary Figure-1: PGK1 leads to β-catenin phosphorylation during inflammation process upon LPS. 50

Appendix B. GST-β-catenin S502A mutation confirmed by sequencing 51

Appendix C. GST-β-catenin T551C mutation confirmed by sequencing 52

Appendix D. GST-β-catenin S552A mutation confirmed by sequencing 53

List of Abbreviations 57

ABSTRACT (in Korean) 59

SUPPLEMENT PUBLICATION 60

Article 61

Table 1. Antibodies were used for this research work 54

Table 2. Primers were used for ChIP-PCR amplification 55

Table 3. Primers were used for Site directed mutagenesis 56

Figure 1. LPS induces superoxide production through RhoA/ROCK, leading to NF-κB activation. (A, B, C) Representative image of cellular ROS/superoxide production... 27

Figure 2. Phosphorylated p65/RelA at Ser536 residue binds with p-Tyr42 RhoA. (A, B) Representative western blotting images depicting the expression levels of RhoA,... 29

Figure 3. p-Ser536 p65/RelA and p-Tyr42 RhoA translocate to nucleus in response to LPS stimulation. (A) HEK293T cells were cultured in a serum-depleted media for 8... 31

Figure 4. LPS induces PGK1 through p-Tyr42 RhoA and p-65. (A) Ven-diagram depicting the intersection of NF-κB and p-Y42RhoA target genes obtained from the... 34

Figure 5. p-Ser536 p65/RelA and p-Tyr42 RhoA bind to promoter of PGK1 gene. (A, B, C) Representative ChIP (Chromatin Immunoprecipitation) data illustrating the... 36

Figure 6. PGK1 leads to β-catenin phosphorylation during inflammation process upon LPS. (A) Cell proliferation was assessed in HEK293T cells transfected with or... 40