ZB4 anti-Fas antibody antagonizes Fas-induced but not ELL-12–induced cell death, DEVDase activity, and caspases-8 and -3 activation in Jurkat cells. (A) Extracts from Jurkat T cells treated with 10 μg/mL ELL-12 for the indicated times were resolved by SDS-PAGE and probed with anti-CD95L antibody. (B) Jurkat T cells were pretreated with 300 ng/mL antagonist anti-Fas MoAb (clone ZB4) for 1 hour and then treated with 50 ng/mL anti-Fas MoAb or 10 μg/mL ELL-12 for an additional 5 hours. Apoptosis was assessed by Hoechst staining. Results are the means ± SD of 3 independent experiments. (C) Activation of DEVD-specific caspases was measured by the cleavage of the fluorogenic substrate Ac-DEVD-AMC. Results are the means ± SD of 3 independent experiments. Cytosolic extracts were subjected to 15% SDS-PAGE and immunoblotted with anti–caspase-8 (D) or anti–caspase-3 (E). (−) and (+) indicate cells treated without or with 300 ng/mL antagonist anti-Fas MoAb (clone ZB4). The migrations indicated are full-length caspase-8, the cleavage intermediates p43 and p41, the active subunit p18, full-length caspase-3, the cleavage intermediate p20, and the active subunit p17.