Figure 6.

Analysis of OxPAPC-induced Src phosphorylation and effects of Src and ROS inhibitors on VE-cadherin phosphorylation and the formation of VE-cadherin–p120-catenin–β-catenin complexes. (A) HPAECs were stimulated with OxPAPC (10 μg/ml or 100 μg/ml) for indicated periods of time, and Src phosphorylation at Tyr⁴¹⁸ and Tyr⁵²⁹ was examined by Western blot analysis. Staining with β-actin antibody was used as a normalization control. (B–D) Cells pretreated with vehicle, Src inhibitor PP2 (1μM), or the antioxidant N-acetyl-cysteine (NAC; 1 mM) for 30 minutes were stimulated with OxPAPC (100 μg/ml, 15 minutes). Concentrations of Src phosphorylation at Tyr⁴¹⁸ were detected by Western blot analysis. (B) Staining with β-actin antibody was used as a normalization control. (C) The assay of ROS production was performed as described in Materials and Methods. (D) Concentrations of VE-cadherin phosphorylated at Tyr⁷³¹ were detected by Western blot analysis. (E) Cells pretreated with vehicle, PP2 (1 μM), or NAC (1 mM) for 30 minutes were stimulated with OxPAPC (100 μg/ml, 15 minutes), followed by immunoprecipitation with anti–VE-cadherin antibody. Coimmunoprecipiated p120-catenin and β-catenin were detected by Western blot analysis with corresponding antibodies. The detection of immunoprecipiated VE-cadherin was used as a normalization control. The results shown are representative of five independent experiments.