The role of src-kinases in PF-4/TNF-mediated exocytosis. (A) Effect of src-kinase inhibitor PP1 or (B) Lyn inhibitor SU6656 on PF-4/TNF-induced lactoferrin release in human neutrophils. Neutrophils were preincubated for 15 minutes in the presence or absence of increasing concentrations of PP1or SU6656, respectively, followed by stimulation with a combination of PF-4 (4 μM) and TNF (9 ng/mL) for 30 minutes at 37°C. Cells that received stimulus alone and were left without inhibitor served as controls. Lactoferrin release was determined in cell supernatants by the use of a sandwich ELISA system. Assay backgrounds (PP1, 1.5% ± 0.1%; or SU6656, 3.1% ± 1.5%, respectively) determined in samples of unstimulated cells run in parallel were subtracted. Data represent mean ± SD of 4 or 3 independent experiments, each performed in duplicate. Asterisk (*) indicates significant differences (PP1, P < .0005; SU6656, P < .007) between inhibitor-treated and untreated samples based on the data from 4 or 3 individual experiments. (C) Activation of srckinases in PF-4– or TNF-stimulated neutrophils. Neutrophils were stimulated with PF-4 (4 μM) or TNF (9 ng/mL) for the time periods indicated and src-kinases were immunoprecipitated (IP) with anti-Lyn, anti-Hck, or anti-Fgr antibodies, respectively, followed by an in vitro phosphorylation assay using acid-denaturated enolase as exogenous substrate. Enolase phosphorylation was visualized using a PhosphorImager system following separation of proteins by SDS-PAGE. (D) Time course of lactoferrin release in response to PF-4/TNF stimulation. Neutrophils were stimulated with a combination of PF-4 (4 μM) and TNF (9 ng/mL) for the times indicated. Lactoferrin release was determined in cell supernatants by the use of a sandwich ELISA system. Assay backgrounds (1.5% ± 0.1%) determined in samples of unstimulated cells run in parallel were subtracted. Data represent mean ± SD of 6 independent experiments, each performed in duplicate.