Fig. 1. Phosphorylation and activation of p38 and JNK precedes caspase-8 induction in Fas-mediated apoptosis in Jurkat cells. Following incubation with Fas-mAb, cells were either stained with annexin-V to detect apoptosis or were lysed and subjected to immunoblot analysis. (A) Fas-induced apoptosis was detectable by 4 hours, increased with time and was maximal by 24 hours. (B) Time-dependent increase occurred in the phosphorylation of p38 (blot 1) and JNK (blot 3) in the absence of any change in the total levels of p38 (blot 2) and JNK (blot 4). (C) The in vitro activity of each kinase was assessed following immunoprecipitation with the respective phosphospecific antibodies. Increased phosphorylation of p38 correlated with its ability to phosphorylate ATF-2 in vitro (blot 1) and of JNK with its ability to phosphorylate JUN (blot 2). (D) The formation of significant amounts of cleaved fragments of caspase-8 (p44/43) and p16 was detectable only beyond 2 hours.