PF4 induced time-dependent activation of Syk. Neutrophils were stimulated for the indicated time periods, and Syk was immunoprecipitated from cell lysates. Precipitates were tested for phosphorylated Syk (pSyk; top panel) and Syk enzyme activity in an in vitro phosphorylation assay using MBP as exogenous substrate (middle panel). Phosphorylation of proteins was detected by Western blot analysis using antibodies directed against phosphotyrosine. IgH indicates immunoglobulin heavy chain. The same lysates were probed with anti-Syk antibody to confirm equal protein loading (bottom panel). Bands were visualized by Odyssey infrared imaging system. Data from 1 of 4 representative experiments are given.