Figure 4.

H₂O₂ induces Gα13 activation and interaction of Gα13 with Src. (A) Confluent HMVEC-L cells were stimulated by multiple inflammatory mediators and H₂O₂ production was assessed 1 h later. Mean ± SD (n = 3 in each group). Results are representative of two experiments. (B) HMVEC-L cell membrane fraction was extracted by CHAPS buffer and treated with trypsin in the presence of varying concentration of H₂O₂ and 50 µM GTP-γS. GTP-γS–loaded Gα13 was protected from digestion by trypsin. Results are representative of two experiments. (C) Serum-starved HMVEC-L cells were treated by 10 mM of the general ROS scavenger NAC for 30 min, and then challenged by 100 µM H₂O₂ at different times. Cell lysates were immunoprecipitated by control IgG or anti-Gα13 antibody, and immunoprecipitates were blotted with antibodies against Gα13 and Src. Results are representative of two experiments. (D) Serum-starved HMVEC-L cells were treated by 10 mM ROS scavenger NAC or 100 µM of the general NOX inhibitor DPI for 30 min, and then stimulated with 20 ng/ml VEGF at different times. Cell lysates were immunoprecipitated by control IgG or anti-Gα13 antibody, and immunoprecipitates were blotted with antibodies against Gα13 and Src. Results are representative of two experiments.