Affinity labeling of active caspases in neutrophils. (A) Time course of caspase activation. Cytoplasmic extracts were obtained from 1 × 10⁷ neutrophils treated with TNF- plus cycloheximide (TNF/CHX) for the indicated time periods (lanes 1 through 4 and 6) or from 5 × 10⁶ Jurkat cells stimulated with anti-Fas antibody (CH-11, 100 ng/mL) for 3 hours (lane 5). Extracts were incubated with (lanes 1 through 5) or without (lane 6) 1 μmol/L zEK(bio)D-aomk for 5 minutes at 37°C. (B) Preferential competition of zEK(bio)D-aomk binding to caspase-3 by DQTD-CHO. Cytoplasmic extracts from 1 × 10⁷ neutrophils treated with TNF- plus cycloheximide for 3 hours were preincubated with DQTD-CHO for 15 minutes at 37°C before labeling with 1 μmol/L zEK(bio)D-aomk. (C) zVAD-fmk inhibition of caspase activation. Neutrophils preincubated for 1 hour with indicated concentrations of zVAD-fmk were stimulated with TNF- plus cycloheximide (lanes 2 through 5) or left untreated (lane 1) for 3 hours. (Upper lanes) zEK(bio)D-aomk–labeled caspases were detected with HRP-conjugated streptavidin. (Lower lanes) The same blot was reprobed with a rabbit anti–caspase-3 antibody. (D) Sensitivities of active caspases to zVAD-fmk. zVAD-fmk at indicated concentrations was added to neutrophils stimulated with TNF- plus cycloheximide for 3 hours. After incubation for 1 hour at 37°C, cytoplasmic extracts were prepared for affinity labeling analysis. (E) Cell-free activation of endogenous caspases in neutrophil extracts by recombinant caspase-8 and by cytochrome c. Cytoplasmic extracts from neutrophils were treated with cytochrome c plus dATP (lanes 1 through 3) or recombinant caspase-8 (lanes 4 through 6) for the indicated time period. Lane 7, cytoplasmic extracts from Fas-stimulated Jurkat cells. All samples were affinity labeled with 1 μmol/L zEK(bio)D-aomk.