Effects of an agonistic anti-Fas/CD95 antibody on PKCδ proteolysis and neutrophil apoptosis. (A) Neutrophils were incubated in the absence (control) or presence of agonistic Fas/CD95 antibody (500 ng/mL), PKC inhibitor, GF109203X (1 μmol/L), and caspase-8 inhibitor, IETD.fmk (25 μmol/L) and cell survival measured by annexin V binding using a flow cytometric assay. Mean ± SE of three separate experiments. (B) Neutrophils were incubated with agonistic Fas/CD95 antibody (500 ng/mL) with or without preincubation with the caspase-8 inhibitor, IETD.fmk (25 μmol/L) for the indicated times. Lysates were prepared, equal amounts of protein (20 μg) loaded per lane, and immunoblotted with an anti–C-terminal PKCδ antibody. (C) Neutrophils were incubated with agonistic Fas/CD95 antibody (500 ng/mL) for the indicated times. Lysates were prepared, equal amounts of protein (20 μg) loaded per lane, and immunoblotted with anti–C-terminal antibodies to PKC or PKCβ.