Angiotensin II–induced JNK1/2 activation in human neutrophils and its dependence on ROS and tyrosine kinase activity. Neutrophils (9 × 106 cells/mL) were suspended in KR-HEPES buffer and preincubated for 7 hours at 37°C without additions. Further, they were incubated in the presence of Ang II (100 nM) for the indicated times (A) or of the indicated Ang II doses for 15 minutes (B), or in the absence or presence of HMAP (500 μM) for 30 minutes or genistein (Gen) (100 μM) for 10 minutes and then with Ang II (100 nM) for 15 minutes (C). After cell lysis, protein extracts (50 μg/lane) were subjected to SDS-PAGE, transferred to PVDF membranes, and probed with antiphospho-JNK1/2. Arrows indicate phosphorylated JNK1 (p46) and JNK2 (p54). To verify even protein loading, the blots were subsequently stripped and reprobed with anti-JNK1/2 antibody. Data are representative of 3 independent experiments that yielded similar results.
