Figure 1

Src Activity coprecipitates with KDR/Flk-1. (A) HUVECs were either serum-starved (-), or starved and then VEGF-treated (15 ng/mL VEGF for 15 min.) (+), or lysed (unstarved, untreated) at subconfluence (C). Cell extracts of these samples were immunoprecipitated using α-Flt-1 (H-225), α-KDR/Flk-1 (N-931), or rabbit nonimmune IgG. Kinase assay reactions were subsequently performed using enolase substrate. Samples were resolved on 10% SDS-PAGE gels. The gels were treated with 1 M KOH at 55°C for 1 h after electrophoresis to remove background due to serine phosphorylation. The gels were then dried and exposed to autoradiographic film (upper panel). (B) Cell extracts from the indicated treatment conditions were immunoprecipitated using α-Flt-1 (H-225) and immunoblotted using α-phosphotyrosine (4G10) or α-Flt-1 (H-225) (left panel); or the cell extracts were immunoprecipitated using α-KDR/Flk-1 (A-3) and immunoblotted using α-phosphotyrosine (4G10) or α-KDR/Flk-1 (N-931) (right panel).