JNK kinase activity is TAK1-dependent. (a) Kinase assays were used to evaluate JNK function in TAK1 deficient cells. Fibroblast-like synoviocytes (FLSs) transfected with TAK1 or scrambled control (sc) small interfering RNA (siRNA) were serum-starved, stimulated with IL-1β (2 ng/ml) for 15 minutes, lysed, immunoprecipitated with anti-JNK antibodies, and subjected to kinase assay using GST-c-Jun substrate. JNK-mediated activation of c-Jun was significantly decreased in TAK1 deficient FLS (53 ± 2% inhibition, p = 0.03). A representative experiment is shown (n = 3). Med, medium; IP, immunoprecipitation. (b) Kinase assays were performed with anti-MKK4 and anti-MKK7 antibody immunoprecipitates and GST-c-Jun substrate to evaluate the effects of TAK1 deficiency on MKK4 and MKK7 function. Significant decreases in IL-1β-induced MKK4 and MKK7 kinase activity were observed (average inhibition: MKK4, 28 ± 4%, p = 0.004; MKK7, 28 ± 8%, p = 0.02). A representative experiment is shown (n = 3). Wb, Western blot.