Fig. 9. p38, but not p38ß is essential for maintaining c-FLIP_S in non-phosphorylated state. Phosphorylation of c-FLIP_S was determined by Pro Q diamond phosphoprotein gel stain following immunoprecipitation with anti-c-FLIP antibody. Phosphorylation of c-FLIP_S is enhanced in si-p38, but inhibited in si-p38ß or si-JNK1/2 cells compared with that seen in native Jurkat cells (top panel). No change in the total c-FLIP_S levels was observed in any of the cells (bottom panel). (B) Jurkat cells expressing control (si-C) or p38 siRNA (si-p38) and flag-p38 or flag-p38-AGF were metabolically labeled with sodium ortho[³²P]phosphate. Phosphorylated c-FLIP was immunoprecipitated from the lysates with anti-c-FLIP antibody that recognizes both c-FLIP_L and c-FLIP_S, electrophoresed on SDS-polyacrylamide gels and subjected to autoradiographic analysis. Phosphorylation of c-FLIP_S was higher in (si-p38, lane 4) than in cells expressing the dominant negative mutant flag-p38-AGF (lane 3) whereas minimal phosphorylation was seen in native Jurkat (lane 1) si-C (lane 2) and Flag-p38 (lane 5) cells. By contrast, c-FLIP_L phosphorylation was unaffected by p38.