Complementary roles of JNK1 and other JNK isoforms in platelet aggregation, TXA₂ synthesis, and PKC activity. The levels of JNK2 expression (A) and phosphorylation (B) were evaluated by Western blotting with appropriate antibodies directed against total JNK2 or phosphorylated JNK2 for WT and JNK1^−/− platelets (10⁸) stimulated with collagen (1 μg/mL) for 3 minutes, with stirring. Tubulin was used as a loading control. The Western blot shown is representative of at least 3 independent experiments. The complementary roles of JNK1 and the other JNK isoforms were evaluated for platelet aggregation (C), TXA₂ synthesis (measured by evaluating production of TXB₂ (D), and PKC activity (by Western blotting with an antibody directed against phosphorylated PKC substrates; E), in WT or JNK1^−/− platelets (10⁸), incubated for 10 minutes at 37°C with DMSO (0.1%, vol/vol), as a control, or with the pan-JNK inhibitor SP600125 (10μM), before stimulation with collagen (1 μg/mL) for 3 minutes, with stirring. The traces and Western blot shown are representative of at least 3 independent experiments. Thromboxane production was assessed by analysis of the supernatants of aggregated platelets, as described in “Methods.” Each bar represents the mean plus or minus SEM of 3 experiments. ***P < .001 (unpaired Student t test).