Fig 4. ARF6 mediates ROS-dependent EGFR transactivation.

(A, B) Control and ARF6 depleted VSMC were stimulated with Ang II (100 nM) for the indicated times. EGFR phosphorylation levels on Tyr¹⁰⁸⁶ (A) and Tyr⁸⁴⁵ (B) were assessed by Western blot analysis using specific phospho antibodies and data normalized to total EGFR (n = 3, *P < 0.05, **P < 0.01). (C) Control and Rac1 T¹⁷N overexpressing VSMC were stimulated or not with Ang II for the indicated times and EGFR phosphorylation levels on Tyr1086 were determined by Western blot analysis (n = 3, **P < 0.01, ***P < 0.001). (D) VSMC were pre-incubated with vehicle or DPI (10 μM) and stimulated with Ang II (100 nM) for the indicated times. EGFR phosphorylation levels on Tyr¹⁰⁸⁶ and Tyr⁸⁴⁵ were determined as in (A, B). Data are the mean ± SEM of three independent experiments (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). (E) VSMC were pre-incubated with vehicle, AG1478 (100nM) or ML171 (5 μM) for 30 min then stimulated with Ang II (100 nM) for the indicated times. EGFR phosphorylation levels on Tyr¹⁰⁸⁶ were measured as in (A) (n = 3,***P < 0.001).