Ligand binding
To determine whether the ASV bound their cognate ligands, 96-well assay plates were coated with VEGF-A, VEGF-C, platelet-derived growth factor (PDGF)-AB, hepatocyte growth factor, colony-stimulating factor (CSF), and Ang-1, respectively, at 4 μg/ml in PBS. The immobilized ligand-coated plates were used for binding of matched ASV in the same order, as follows: VEGFR1-541, VEGFR2-712, PDGFRβ-336, Met-877, CSF1R-306, and Tie1-751. In the case of VEGFR1-541, VEGFR2-712, PDGFRβ-336, Met-877, and CSF1R-306, supernatants from the ASV-expressing HEK293 cells were used for binding assays. The purified Tie1-751(6His) was used for Ang-1 binding. Binding was performed for 1.5 hours at room temperature followed by three rapid rinses in PBS/0.05% Tween-20. Bound ASV were detected using biotin-labeled, extracellular domain-specific antibodies.