15(S)-HETE induces αPix tyrosine phosphorylation in Src-dependent manner. (A) Quiescent HDMVECs were treated with and without 0.1μM 15(S)-HETE for the indicated time periods and αPix tyrosine phosphorylation was measured by immunoprecipitation with anti-αPix antibodies followed by immunoblotting with anti-PY20 antibodies. (B) Cells were transduced with Ad-GFP or Ad-dnSrc, growth-arrested, treated with and without 0.1μM 15(S)-HETE for 10 minutes and analyzed for αPix tyrosine phosphorylation as described in panel A. (C) HDMVECs were transfected with scrambled (Scr) or αPix siRNA and 48 hours later cell extracts were prepared and analyzed for αPix levels by Western blotting using its specific antibodies. (D) HDMVECs that were transfected with scrambled or αPix siRNA and growth-arrested were treated with and without 0.1μM 15(S)-HETE for 1 hour and Rac1 activation was measured. (E-F) All the conditions were the same as in panel D except that cells were subjected to 0.1μM 15(S)-HETE-induced migration (E) or tube formation (F). The bar graphs represent the mean ± SD values of 3 independent experiments. *P < .01 versus control; **P < .01 versus 15(S)-HETE.