15(S)-HETE–induced FGF-2 expression is mediated by Src-Egr-1 signaling in HDMVECs. (A-B) HDMVECs were transduced with Ad-GFP, Ad-dnSrc, or Ad-dnEgr-1 with a moi of 40, quiesced, and treated with and without 15(S)-HETE (0.1μM) for either 30 minutes for RNA isolation or 30 minutes and/or 60 minutes for cell extract preparation. RNA was analyzed by QRT-PCR for FGF-2 and β-actin mRNA levels, and cell extracts were analyzed by Western blotting for FGF-2 levels with its specific antibodies. The blots were reprobed with anti–β-tubulin antibodies and/or anti-Src antibodies or anti-WT1 antibodies for normalization and to show overexpression of dnSrc or dnEgr-1, respectively. (C) All the conditions were the same as in panel A except that medium was collected and tested for FGF-2 release by ELISA. The bar graphs in panels A and C represent the quantitative analysis of 3 independent experiments. The values are presented as the means ± SD. *P < .01 vs Ad-GFP; **P < .01 vs Ad-GFP + 15(S)-HETE.