Title Page
Contents
ABSTRACT 8
Ⅰ. Introduction 10
Ⅱ. Material and Methods 12
2.1. Expression of human IL-32θ mutant (A94V) 12
2.2. Purification of IL-32θ mutant (A94V) using Ni-NTA and CNBr-activated- 3 -Sepharose 4B columns 12
2.3. 3D structure modeling of IL-32θ mutant (A94V) and IL-32θ mutant (A94V)-integrin binding prediction 13
2.4. IL-32θ mutant (A94V)-integrin binding assay 14
2.5. Cell culture 14
2.6. RNA extraction and reverse-transcription polymerase chain reaction (RT-PCR) 15
2.7. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) 15
2.8. Immunoblotting 16
2.9. Immunofluorescence 17
2.10. siRNA transfection 17
2.11. Monocyte-endothelial cell adhesion assay 18
2.12. Preparation of cytosol and nuclear extracts 18
Ⅲ. Results 19
3.1. Expression and purification of recombinant human IL-32θ mutant (A94V) 19
3.2. Binding of IL-32θ mutant (A94V) to integrin αVβ3 and integrin αVβ6 21
3.3. Effects of IL-32θ mutant (A94V) on expression of VCAM-1 and ICAM-1 in TNF-α-stimulated HUVEC cells 24
3.4. Effects of IL-32θ mutant (A94V) on monocyte-endothelial cell adhesion 26
3.5. Effects of IL-32θ mutant (A94V) on phosphorylation levels of FAK, AKT, and JNK in TNF-α-stimulated HUVEC cells 29
3.6. Effects of IL-32θ mutant (A94V) on nuclear translocation of NF-κB and AP-1 in TNF-α-stimulated HUVEC cells 30
Ⅳ. Discussion 32
References 37
Abstract (in Korean) 43
Figure 1. Expression and purification of IL-32θ mutant (A94V). (A) Schematic diagram of mutant IL-32θ mutant (A94V) expression vector. As a protein expression... 20
Figure 2. The tertiary structure modeling of IL-32θ mutant (A94V) and prediction of IL-32θ mutant (A94V)-integrin binding. Protein-protein interactions were predicted... 22
Figure 3. IL-32θ mutant (A94V) binding to integrin αVβ3 and integrin αVβ6. (A) IL-32θ mutant (A94V) binds to the recombinant αVβ3 and αVβ6 integrins. The effects... 23
Figure 4. The effect of IL-32θ mutant (A94V) on mRNA expression of VCAM-1 and ICAM-1 in TNF-α stimulated HUVECs. (A) The expression of integrin subunits, αV,... 25
Figure 5. The effect of IL-32θ mutant (A94V) on protein expression of VCAM-1 and ICAM-1 in TNF-α stimulated HUVECs. HUVECs were pre-treated with IL-32θ... 26
Figure 6. IL-32θ mutant (A94V) attenuates monocyte adhesion to HUVECs. (A) THP-1 cells were treated with IL-32θ mutant (A94V) (100 ng/ml) for 24 hours. LFA-... 28
Figure 7. Effect of IL-32θ mutant (A94V) on phosphorylation level of FAK, JNK, and AKT in HUVEC cells. HUVECs were pre-treated with IL-32θ mutant (A94V)... 29
Figure 8. The effect of IL-32θ mutant (A94V) on phosphorylation levels of IκB and nuclear translocation of NF-κB, AP-1 in HUVEC cells. HUVEC cells were incubated... 31
Figure 9. Schematic diagram of the effect of IL-32θ mutant (A94V) on the TNF-α- induced VCAM-1 and ICAM-1 expression signaling pathway. TNF-α-induced... 35