VEGFR signaling via Rac1/ROS/Pyk2 triggers VE-cadherin/VE-PTP dissociation. (A) Rac1 inhibition prevents VEGF-induced dissociation. bEnd.5 cells were treated with the Rac1 inhibitor NSC23766 (25 μM) before incubation with VEGF, followed by VE-PTP/VE-cadherin coimmunoprecipitation analysis. (B) NOX inhibition prevents VEGF-induced dissociation. bEnd.5 cells were treated with the antioxidant NAC (1 mM) or the NOX inhibitor VAS2870 (10 μM) before incubation with VEGF, followed by VE-PTP/VE-cadherin coimmunoprecipitation analysis. (C) Pyk2 activity is required for VEGF-induced dissociation. bEnd.5 cells were pretreated with the dual Pyk2/FAK inhibitor PF431396 (5 μM) before incubation with VEGF, followed by VE-PTP/VE-cadherin coimmunoprecipitation analysis. (D) VEGF-triggered activation of Pyk2 requires NOX activity. bEnd.5 cells were pretreated with the ROS scavenger NAC (1 mM) or the NOX inhibitor VAS2870 (10 μM) before incubation with VEGF, followed by immunoprecipitating Pyk2 and immunoblotting for phospho-Pyk2 (WB: phospho-Pyk2 total lysate). Equivalent aliquots of total cell lysates were analyzed by immunoblotting for Pyk2 (WB: Pyk2 total lysate).