Figure 5

Monitoring activation of NF-κB. (a) Rabbit synovial fibroblast HIG-82 cells were transiently transfected with pNFκB-MetLuc2-Reporter Vector (Clontech). The transfected cells were stimulated with IL-1β (10 ng/ml), in the presence and absence of either IDR-1002 or IDR-1. The activation of NF-κB was monitored after 6 hours of stimulation by using Ready-To-Glow Secreted NF-κB Luciferase Reporter Assay (Clontech) as per the manufacturer's instructions. Results shown are an average of at least three independent experiments ± standard error (*P < 0.05; NS, nonsignificant). (b) Nuclear extracts of human fibroblast-like synoviocytes (FLS) stimulated with IL-1β (10 ng/ml) in the presence and absence of either IDR-1002 or IDR-1 were probed with NF-κB p50 antibody or β-actin antibody in immunoblots. IDR-1002 was added either (i) 45 minutes before, or (ii) at the time of cytokine stimulation. Result shown is a representative blot of three independent experiments performed with FLS obtained from three different donors. IDR, innate defence regulator; IL, interleukin; NF-κB, nuclear factor-kappaB.