VEGF-induced PKCγ activation is mediated by PLD1 in HRMVECs. (A) Quiescent HRMVECs were treated with and without VEGF (40 ng/mL) for the indicated time periods, and cell extracts were prepared and analyzed by Western blotting for PKCα/βII, -γ, and -δ phosphorylation using their phosphospecific antibodies. (B) All the conditions were the same as in panel A, except that cells were treated with and without VEGF (40 ng/mL) in the presence and absence of 1-butanol (0.25%) or propranolol (200μM) for 30 minutes and cell extracts were prepared and analyzed by Western blotting for PKCα/βII, -γ, and -δ phosphorylation using their phosphospecific antibodies. (C) HRMVECs that were transfected with scrambled or PLD1 siRNA were quiesced, treated with and without VEGF (40 ng/mL) for 30 minutes, and cell extracts were prepared and analyzed by Western blotting for PKCγ phosphorylation using its phosphospecific antibodies. (A-C) The blots were reprobed with the antibodies of the indicated molecules for either normalization or to show the effect of siRNA on its target molecule levels.