Src mediates VEGF-induced PLD1-PKCγ signaling axis activation. (A) All the conditions were the same as in Figure 1A, except that the cell extracts were analyzed by Western blotting for Src phosphorylation using its phosphotyrosine antibodies. (B) Quiescent HRMVECs were treated with and without VEGF (40 ng/mL) for 30 minutes in the presence or absence of 10μM PP1, a potent Src inhibitor, and cell extracts were prepared and analyzed by Western blotting for PLD1 and PKCγ phosphorylation using their phosphospecific antibodies. (C) All the conditions were the same as in panel B, except that cells were transduced with Ad-GFP or Ad-dnSrc at 40 multiplicity of infection and quiesced before subjecting them to treatment with and without VEGF (40 ng/mL) for 30 minutes and analyzing the cell extracts for PLD1 and PKCγ phosphorylation. (A-C) The blots were reprobed with antibodies of the indicated molecules for normalization. The bottom blot in panel C shows overexpression of dominant negative Src. (D-F) Quiescent cells in which Src is inhibited by PP1 or its dominant negative mutant were treated with and without VEGF (40 ng/mL), and DNA synthesis, cell migration, and tube formation were measured as described in Figure 1B, C, and D, respectively. Values are mean ± SD. *P < .01 versus vehicle control or Ad-GFP control. **P < .01 versus VEGF or Ad-GFP plus VEGF.