Fig. 4

Regulation of semaphorin 4A (Sema4A) expression by NF-κB. a Schematic illustration as the binding of NF-κB to the promoter of Sema4A. b Synovial fibroblasts of rheumatoid arthritis (RASFs) and THP-1 cells were transfected with 50 nM of control, p50 or p65-siRNA, or p50 and p65 combined and incubated for the indicated time. Total extracts were separated by SDS-PAGE and western blot analysis was conducted for measurement of Sema4A protein levels. c Chromatin immunoprecipitation (ChIP) analysis showed that p65 was recruited to the Sema4A promoter after treatment with lipopolysaccharide (LPS) for 90 minutes. On ChIP analysis with control primers against distinct promoter regions there was no recruitment of p65. Input, chromatin input before immunoprecipitation; Anti-p65, immunoprecipitated chromatin with anti-p65 antibody; IgG, immunoprecipitated chromatin with control IgG. Effect of NF-κB inhibitor treatment on the LPS-induced Sema4A was detected by qRT-PCR (d) and western blot (e)