VEGFR-1 dII mediates cell adhesion and interaction with α5β1 integrin. (A) In a solid-phase binding assay, plates coated with VEGFR-1/Fc were treated with anti–VEGFR-1 antibodies and incubated with purified α5β1 integrin. The amount of bound integrin was quantified by incubation with an anti-α5β1 antibody and colorimetric detection of this antibody. (B) HUVECs were incubated on wells coated with recombinant dII. VEGFR-1/Fc and fibronectin (FN) were used as positive controls, bovine serum albumin (BSA) as a negative control. The relative number of attached cells was assessed by staining with crystal violet and determining A₅₄₀ 1 hour after plating. Absorbance resulting from nonspecific cell adhesion was measured on BSA-coated wells. (C) Purified VEGFR-1/Fc or dII were added at the indicated concentrations to wells coated with α5β1 integrin. Bound molecules were detected using either an anti-Fc alkaline phosphatase (AP)-conjugate or an anti–VEGFR-1 antibody and a secondary anti-rabbit antibody AP-conjugate. Representative experiments performed in triplicate are shown; data are mean plus or minus SEM.