TNF-α-induced p38 MAPK activity is impaired in rip1−/− cells. (A) Wild-type and rip−/− MEF were grown to confluence on 10-cm-diameter plates and treated with 10 ng of murine TNF-α/ml for 0, 10, 30, and 60 min. Cells were subsequently lysed and immunoprecipitated with anti-p38α Ab (a gift from R. Davis), and the kinase activity of p38 MAPK was measured by an in vitro kinase assay with GST-ATF2 as substrate. The p38α level in each cell type was determined by immunoblotting with anti-p38α (Santa Cruz). (B) Normal IL-1α-induced p38 MAPK activity in rip−/− cells. Wild-type and rip−/− MEF were treated with 10 ng of IL-1α/ml for various times (0, 10, 30, and 60 min), and the p38 MAPK activity was measured by immunoblotting with an anti-phospho-p38 Ab (Cell Signaling Technology catalog no. 9211S). (C) Normal sorbitol-induced p38 MAPK activity in rip−/− cells. Wild-type and rip−/− MEF were treated with 0, 100, 200, and 400 mM sorbitol for 15 min, and the kinase activity of p38 was measured by in vitro kinase assay with GST-ATF2 as substrate.