Prolonged phosphorylation induced by matrix-bound VEGF₁₆₅ is specific to tyrosine 1214. (A) HUVECs were incubated with Vs or Vb (200 ng/ml) for either 5 or 15 min, as indicated. Phosphorylation assays were performed and 4G10 was used as pan-phosphotyrosine antibody. Densitometric analysis of phospho-VEGFR2 by Vs and Vb were normalized with total VEGFR2 and then compare Vb over Vs as indicated. (B) Phosphorylation assays were performed using phosphotyrosine antibodies specific for the indicated residues on VEGFR2. (C) HEK 293 cell lines expressing Y1214F VEGFR2 mutant were incubated with soluble (Vs; 200 ng/ml) or bound VEGF₁₆₅ (Vb) for 5 min and 15 min, vehicle (−), or collagen only (col) for 5 min. Equal protein levels were separated by gel electrophoresis and probed with 4G10. The blot was then reprobed with VEGFR2 antibody for normalization. Densitometric analysis of phosphorylation status induced by Vb to Vs is indicated. (D) Similar experiments were performed using HEK 293 stably transfected with native VEGFR2 (293NR).