The JAK pathway: a critical pathway for TNF-induced functional caspase-1. Rheumatoid arthritis (RA) synovial fibroblasts (2 × 105/well; 2 ml/well) were pre-incubated with the indicated inhibitors for 2 hours, followed by stimulation with TNF (20 ng/ml) for 48 hours. Caspase-1 was assessed at the mRNA level (A), the protein level (B), and its activity was measured (C). RA synovial fibroblasts (2 × 105/well; 2 ml/well; in RPMI with 10% fetal bovine serum (FBS)) were pre-incubated with AG490 for 2 hours, followed by stimulation with TNF for 48 hours. Medium was collected and concentrated, while cell lysates were processed. IL-18 levels were assessed by ELISA in both conditioned media (D) and cell lysates (E). IL-18BP levels (F) and IL-18 bioactivity (G) were assessed in the conditioned media. Bars show the mean ± standard error of the mean (SEM). *P <0.05. NS, non stimulated; n, number of donors and independent experiments.