Prolonged activation of VEGFR2 requires matrix-binding motifs in VEGF. (A) Various VEGF isoforms (165, 121, 113; 200 ng/ml) were polymerized with collagen as indicated. The gel was prepared and washed as described in Materials and methods and placed on confluent monolayers for 5 min. Phosphorylation assays were performed and total VEGFR2 levels were determined by reprobing the same membrane. (B) Same as A, but treated for 15 min and probed with antibodies against pY1214 (top) or pP38 (bottom). Numbers on the bottom of the Western blot indicate densitometric quantification. (C) Different collagen types were used to anchor VEGF (Vb, 200 ng/ml) as indicated. Exposure to collagen matrices for 15 min resulted in prolonged phosphorylation of Y1214. (D) Dose curve of VEGFR2 activation by VEGF. Detection of phosphorylation was performed using 4G10 antibodies. Numbers below the Western blotndicate densitometric quantification at 15 min using 100 ng/ml VEGF₁₆₅ treatment as 1.0. (E) 500 ng/ml of VEGF₁₆₅ was used on cells for 5, 15, and 30 min in the presence or absence of a carrier to stabilize the protein. On the right, 500 ng/ml of VEGF was used at time 0, and an additional fresh aliquot at 500 ng/ml was added at indicated time points for another 5 min. Phosphorylation assay was performed using 4G10 antibody.