Fig. 3
FLIP-specific siRNAs enhance TRAIL-induced processing of caspase-8. SV80 (A) and KB cells (B) (107 cells/ml) were transiently transfected with the indicated siRNAs (150 nM) as described in Figure 2. After the second electroporation, cells were seeded in 2 ml of medium in 6-well plates. The next, day cells were challenged for 4 hr with anti-Flag (1 µg/ml) cross-linked TRAIL (100 ng/ml). Finally, detached and attached cells were harvested, lysed together in RIPA buffer supplemented with 0.1 vol of a protease inhibitor cocktail stock solution (Roche), and analyzed for procaspase-8 processing by Western blotting using a caspase-8-specific monocolonal antibody (mAb) recognizing the p55/53 procaspase-8 isoforms and the processed p18 subunit. Western blotting with a vimentin-specific mAb served as control for protein loads.