Angiotensin II–induced p38MAPK activation is dependent on ROS production, Ca2+ levels, tyrosine kinase, and Ptx-sensitive G proteins. Neutrophils (9 × 106 cells/mL) were incubated previously for 7 hours in KR-HEPES at 37°C without additions. Further, they were incubated in the absence or presence of DPI (10 μM) (A); HMAP (500 μM) or AESBF (500 μM) for 30 minutes (B); NAC (50 mM) or PDTC (100 μM) for 60 minutes (C); aminotriazole (D); TEMPO (100 μM) or SOD (500 U/mL) for 30 minutes (E); EGTA (2.5 mM) for 30 minutes (F); herbimycin A (Herb A) (3 μM) for 30 minutes (G); genistein (Gen) (100 μM) for 10 minutes or pertussis toxin (Ptx) (10 ng/mL) for 2 hours (H); and wortmannin (Wort) (100 nM) for 30 minutes (I). Cells were then treated with angiotensin II (Ang) at the indicated concentrations, or at 100 nM (D-E), for 30 minutes and subsequently lysed. Protein extracts (50 μg/lane) were subjected to SDS-PAGE, transferred to PVDF membrane, and probed with antiphospho-p38MAPK. To verify even protein loading, the blots were subsequently stripped and reprobed with anti-p38 antibody (A-C, E-H). Results shown in all panels are representative of 3 independent experiments.
