Evaluation of JNK and p38 MAPK activation. (a) Human fibroblast-like synoviocytes (FLS) were stimulated with IL-1β (10 ng/ml) in the presence and absence of IDR-1002 for 15 minutes. Immunoprecipitation (IP) was performed by using 20 μg of cell lysates with a JNK-specific antibody. The IP eluates were incubated with c-Jun substrate and ATP mixture, and the kinase activity specific to JNK was monitored by monitoring the phosphorylation of the substrate by using an anti-phospho-c-Jun (Ser73)-specific antibody. (b) Human FLS were stimulated with IL-1β (10 ng/ml) in the presence and absence of either IDR-1002 or IDR-1 for 15 minutes. The peptides were added either (i) 45 minutes before or (ii) at the time of cytokine stimulation. IP was done by using 10 μg of cell lysates with a p38-specific antibody, and the IP eluates were incubated with substrate ATF-2 protein and ATP mixture. Kinase activity specific to p38 MAPK was evaluated in immunoblots probing with a phospho-ATF-2 (Thr76)-specific antibody. The immunoblots shown are representative of three independent experiments performed with cells isolated from synovial tissues of three different donors. IDR, innate defence regulator; IL, interleukin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP-3, matrix metalloproteinase-3.