VEGFA stimulates Kdr but not Flt1 tyrosine phosphorylation in HRMVECs. (A,B) Quiescent HRMVECs were treated with and without VEGFA (40 ng/mL) for the indicated time periods and cell extracts were prepared. An equal amount of protein from the control and each treatment group was immunoprecipitated with anti-Kdr or anti-Flt1 antibodies and the immunocomplexes were analyzed by western blotting using anti-PY20, anti-Kdr, or anti-Flt1 antibodies. An equal amount of protein from control and each treatment was also analyzed by western blotting for total Kdr and Flt1 levels using their specific antibodies. (C) All the conditions were the same as in panel A except that an equal amount of protein from the control and each treatment group was immunoprecipitated with anti-Flt1 antibodies. The immunocomplexes were analyzed by western blotting using anti-PY20 antibodies, and the blot was reprobed sequentially with anti-Kdr and anti-Flt1 antibodies. The bar graphs represent the quantitative analysis of 3 independent experiments. The values are presented as mean ± SD. *P < .01 vs control. Anti-PY20 antibodies, phosphotyrosine antibodies.