FIG. 4.

Caspase 8 can activate Cif in vitro. (A) Aliquots (100 μg) of S-100 cytosolic extract from Jurkat cells were left untreated (lane 1) or treated with 4 μl (100 ng) of caspase 8 (lane 3) or 4 μl (100 ng) of caspase 8 plus 1 μl of Ac-DEVD-CHO (250 μM) (lane 4) at 37°C for 60 min. Then the samples were incubated with mitochondria for 15 min at 37°C to assay for the presence of Cif activity. Alternatively, the S-100 extract was treated with caspase 8 as in lane 3 and then at the end of the treatment, 10 μM Ac-DEVD-CHO was added to the sample, which was then assayed for Cif activity (lane 5). As an additional control, the mitochondria were incubated with just 25 μl of buffer (no S-100 cytosolic extract) containing 100 ng of caspase 8 (lane 2). At the end of each treatment, the samples were centrifuged to pellet the mitochondria. The pellet (P) and supernatant (S) were separated, and the cytochrome c (Cyt. c) content in each fraction was determined by Western blot analysis. (B) Aliquots (100 μg) of S-100 extract from Jurkat cells were left untreated or treated with 100 ng of caspase 8 at 37°C for 60 min. The samples were then subjected to a Western blot analysis for caspase 3.