FIGURE 3.

Src mediates VEGF-induced cPLA₂ phosphorylation and AA release in HRMVECs. A, quiescent HRMVECs were treated with and without VEGF (40 ng/ml) in the presence or absence of PP1 (10 μm) for 30 min, and cell extracts were prepared and analyzed by Western blotting for cPLA₂ phosphorylation using its phosphospecific antibodies. The blot was reprobed sequentially with anti-cPLA₂ and anti-β-tubulin antibodies for normalization. B, all conditions were the same as in A except that cells were transduced with Ad-GFP or Ad-dnSrc at 40 multiplicities of infection and growth-arrested before subjecting them to treatment with and without VEGF (40 ng/ml) for 30 min and analyzing the cell extracts for cPLA₂ and Src phosphorylation. The blot was reprobed sequentially with anti-cPLA₂, anti-Src, and anti-β-tubulin antibodies for normalization or to show overexpression of dominant negative Src. C, HRMVECs that were prelabeled with [³H]AA and growth-arrested were treated with and without VEGF (40 ng/ml) in the presence and absence of PP1 (10 μm) for 1 h, and [³H]AA released into the medium was measured. D, all conditions were the same as in B except that cells were transduced with Ad-GFP or Ad-dnSrc at 40 multiplicities of infection and prelabeled with [³H]AA before subjecting them to treatment with and without VEGF (40 ng/ml) for 1 h and measuring [³H]AA released into the medium. The values are presented as mean ± S.D. *, p < 0.01 versus vehicle control or Ad-GFP control; **, p < 0.01 versus VEGF or Ad-GFP + VEGF.