VEGFB stimulates Flt1 tyrosine phosphorylation and Src-PLD1-PKCγ-cPLA₂ activation in HRMVECs as well as the migration and tube formation of these cells. (A) Quiescent HRMVECs were treated with and without VEGFB (40 ng/mL) for the indicated time periods and cell extracts were prepared. An equal amount of protein from control and each treatment group was immunoprecipitated with anti-Flt1 or anti-Kdr antibodies and the immunocomplexes were analyzed by western blotting using anti-PY20 antibodies. The blots were reprobed with anti-Flt1 or anti-Kdr antibodies for normalization. (B) All the conditions were the same as in panel A except that the cell extracts consisting of an equal amount of protein from control and each treatment were immunoprecipitated with anti-Flt1 antibodies and the immunocomplexes were immunoblotted with anti-Kdr antibodies. (C) All conditions were the same as in panel A except that an equal amount of protein from control and each treatment group was analyzed for pSrc, pPLD1, pPKCγ, and pcPLA₂ levels using their phosphospecific antibodies. The blots were reprobed with anti-Src, anti-PLD1, anti-PKCγ, anti-cPLA₂, or anti-β-tubulin antibodies for normalization. (D-F) Quiescent HRMVECs were treated with and without VEGFB (40 ng/mL) and subjected to DNA synthesis (D), migration (E), or tube formation (F) as described in the Figure 3 legend. The representative HRMVEC migration and tube formation images are shown in panels E and F, respectively. The bar graphs in panels A and C-F represent the quantitative analysis of 3 independent experiments. The values are presented as mean ± SD. *P < .01 vs control; ^†P < 0.05 vs control.