VEGFR2 associates with β1 integrin when exposed to matrix-bound but not soluble VEGF₁₆₅. (A) Confluent HUVEC monolayers were incubated with soluble (200 ng/ml) or bound VEGF₁₆₅ (200 ng/ml) and collagen for 5, 15, and 30 min. Cultures were then lysed and equal cell lysates were used for immunoprecipitation with VEGFR2 antibodies. The precipitated protein was then resolved on 4–12% SDS-PAGE and probed with a β1 integrin antibody (130 kD). The blot was then reprobed with anti-VEGFR2. (B) Representative images of proximity ligation assays with quantification (C) after treatment with the indicated growth factors. Red dots denote regions of signal amplification consistent with VEGFR2/β1 integrin interactions. Nuclear stain is TOPRO3 (blue) (n = 4; *, P < 0.05 between Vb to either −, Vs, or Col).