Matrix-bound VEGF₁₆₅ is able to phosphorylate VEGFR2. (A) Confluent PAE-KDR cultures were exposed for 5 min to matrix-bound VEGF₁₆₅ (Vb), soluble VEGF₁₆₅ (Vs; 100 ng/ml and 200 ng/ml), vehicle only (−), or polymerized collagen gels. Level of VEGFR2 (210 kD) phosphorylation was determined by Western blot analysis using a pan-phosphotyrosine antibody (4G10). The bottom panels are blots reprobed with antibodies against VEGFR2, as control. (B) Confluent HUVECs, HSVECs, and HAECs were exposed to Vb, Vs, or matrix-collagen gel controls. The blots were stripped and reprobed with antibodies against VEGFR2 as controls. (C) Similar experiment settings as A and B using 200 ng/ml VEGF, but in the presence of a nylon membrane when indicated. (D) VEGFR2 phosphorylation was induced by different concentrations of Vs or Vb, as indicated. (E) VEGF₁₆₅ (200ng/ml) from two different sources was compared under bound and soluble conditions. BB94 (MMP inhibitor, 2 µM) was incorporated within the collagen gel together with VEGF₁₆₅. The arrows indicate different phosphorylation patterns of VEGFR2 caused by Vb treatment. The densitometry of D and E were calculated by p-VEGFR2 over VEGFR2 and using vehicle (−) as 1.0. (F) HUVECs exposed to either soluble (200 ng/ml) or matrix-bound VEGF₁₆₅ and at the indicated times. Phosphorylation levels of VEGFR2 were then determined by Western blot using 4G10 antibody. Note that prolonged phosphorylation of VEGFR2 in bound when compared with soluble VEGF₁₆₅. (G) Densitometric analysis of same experiments as F (n = 4–5), shown as ratio of Vb over Vs in each time point (*, P < 0.05 for 10- and 20-min time point between Vb and Vs).