Role of caspases in PKCδ proteolysis and neutrophil survival. (A) Effect of caspase inhibitors on PKCδ proteolysis in neutrophils. Neutrophils were either lysed immediately after preparation (t0 con) or after 8 hours incubation in the absence (cont) or presence of broad spectrum caspase inhibitor, BD.fmk, the caspase-8 inhibitor, IETD.fmk (both at 25 μmol/L), the caspase-2 inhibitor, VDVAD.fmk (50 μmol/L), or with agonistic Fas antibody (500 ng/mL) as indicated and immunoblotted with anti-PKCδ antibody. (B) Effect of recombinant caspases on PKCδ proteolysis. Purified recombinant caspase-3 or caspase-8 was incubated with recombinant PKCδ ± caspase inhibitor DEVD-CHO and proteolysis estimated by immunoblotting with an anti-PKCδ antibody. For comparison, a lysate from 8-hour aged neutrophils is shown (PMN). (C) Effect of caspase inhibitors on neutrophil survival. Neutrophils were incubated in the absence (cont) or presence of the broad spectrum caspase inhibitor, BD.fmk, the caspase-8 inhibitor, IETD.fmk (both at 25 μmol/L), or the caspase-2 inhibitor, VDVAD.fmk (50 μmol/L) for 20 hours and survival measured by annexin V binding and flow cytometry. Mean ± SE of four separate experiments. (D) Effect of the caspase-8 inhibitor, IETD.fmk, and the PKC inhibitor, GF109203X, on apoptosis induced by an agonistic Fas antibody. Neutrophils were preincubated with IETD.fmk (25 μmol/L) or GF109203X (1 μmol/L) for 15 minutes and then anti-Fas antibody added (500 ng/mL). Cell survival was measured by annexin V binding in a flow cytometric assay. Mean ± SE of three separate experiments.