Measurement of PKC kinase activity in aged neutrophils. (A) Neutrophils were incubated in the absence (t8h) or presence of the caspase-8 inhibitor, IETD.fmk (25 μmol/L), agonistic anti-Fas antibody (500 ng/mL), PKC inhibitor, GF109203X (1 μmol/L), or PKC inhibitor, Gö6976(1 μmol/L) for 8 hours. PKCδ was then immunoprecipitated using an anti–C-terminal antibody from neutrophil lysates and subjected to a kinase assay using histone H1 as a substrate. Due to the competitive, reversible nature of the PKC inhibitors (which would wash out during the immunoprecipitation protocol), further compound was added to a final concentration of 500 nmol/L to the kinase mixture before the addition of ATP. Phosphorylation of substrate was quantified after autoradiography and normalized to the time 0 value (100%). Results are mean ± SE of three separate experiments. (B) Representative autoradiograph from a PKCδ kinase assay. (C) PKCs  and βΙΙ were immunoprecipitated using anti–C-terminal antibodies after neutrophil aging for 8 hours in the absence or presence of the PKC inhibitor, Gö6976, and subjected to a kinase assay using histone H1 as a substrate as detailed above. Mean ± SE of three separate experiments.