MEKK1, MEKK2 and MEKK3 do not alter IL-1β-induced mitogen-activated protein kinase (MAPK) activation. (a) Three days after small interfering RNA (siRNA) transfection, serum-starved fibroblast-like synoviocytes (FLSs) were stimulated with IL-1β (2 ng/ml) for 15 minutes and lysates were evaluated by western blot analysis. MEKK1, MEKK2, or MEKK3 deficiency alone or in combination had no effect on IL-1β-stimulated JNK, p38 or ERK activation compared with scrambled control (sc) (n = 2 separate FLS lines). M1, MEKK1 siRNA; M2, MEKK2 siRNA; M3, MEKK3 siRNA; M123, MEKK1+MEKK2+MEKK3 siRNA. (b) To complement the MEKK1 siRNA studies, MEKK1-/- FLSs were also examined. MEKK1 knockout (KO) and wild-type (WT) mouse FLSs were serum-starved for 48 h, stimulated with IL-1β (2 ng/ml) for 15 minutes and lysed. Cell extracts were evaluated by western blot analysis. MEKK1 deficiency did not affect IL-1β-induced MAPK activation. (c) MEKK1-/- mFLSs were transfected with MEKK2 and MEKK3 (M2M3) or sc siRNA, and, later, serum-starved for 48 h and stimulated with IL-1β (2 ng/ml) for 15 minutes. Western blot analysis of the lysates confirmed that MEKK2 and MEKK3 do not alter MAPK activation.