PF4 induces the phosphorylation and activation of JNK. Neutrophils were stimulated with PF4 (4 μM) for the time periods indicated in the figure. Cell lysates were prepared, and proteins were separated by SDS-PAGE. JNK phosphorylation was detected by Western blot analysis using anti-phospho-JNK antibodies (A). Alternatively, cells were lysed, and enzymatically active JNK was pulled down using c-Jun fusion protein beads. Kinase activity was determined by the phosphorylation of c-Jun fusion protein and visualized by Western blot analysis using an antibody directed against Ser63-phosphorylated c-Jun (B). An aliquot of the same lysates was tested with anti-JNK1 antibodies to confirm equal protein loading (C). Bands were visualized by the Odyssey infrared imaging system. Data from 1 of 4 representative experiments are given.