14,15-EET–induced VEGF expression is mediated by Src-STAT-3 signaling in HDMVECs. (A,B) HDMVECs were transduced with Ad-GFP, Ad-dnSrc, or Ad-dnSTAT-3 with an MOI of 80, quiesced, and treated with and without 14,15-EET (0.1 μM) for 2 hours, and RNA was isolated and analyzed for VEGF mRNA levels by RT-PCR (A), or for 6 hours, and the VEGF release into the culture medium was measured by ELISA (B). (C,E) Quiescent HDMVECs were treated with and without 14,15-EET (0.1 μM) for the indicated time periods and nuclear extracts were either prepared and analyzed by EMSA for STAT binding using [³²P]-labeled STAT consensus sequence of VEGF promoter as a probe in vitro (C) or processed for ChIP analysis of STAT-3 binding to VEGF promoter in vivo (E). (D,F) HDMVECs that were transduced with Ad-GFP, Ad-dnSrc, or Ad-dnSTAT-3 with an MOI of 80 and quiesced were treated with and without 14,15-EET (0.1 μM) either for 30 minutes and nuclear extracts were prepared and analyzed by EMSA for STAT binding as described in panel C (D) or for 1 hour and processed for ChIP analysis of STAT-3 binding to VEGF promoter in vivo (F). The values in the bar graph in panel B are the means plus or minus SD of 3 independent experiments. *P < .01 vs Ad-GFP; **P < .01 vs AD-GFP + 14,15-EET.