Detection of FGF2/LYVE-1 interaction using other assays. (A) Interaction of LYVE-1 with various ligands detected by a solid-phase ligand-binding assay. LYVE-1 was added to wells coated with different recombinant proteins as indicated, and incubated at 2 hours at 37°C. Anti–LYVE-1, secondary peroxidase-conjugated anti–goat antibodies, and TMB substrate were used to detect bound LYVE-1. Representative experiment was done in duplicate. Error bars represent the mean ± SD (n = 4). Values of dissociation constants (KD) are presented in Table in the bottom. (B) Cross-linking of FGF2 to LYVE-1 in solution. FGF2 labeled with near-infrared fluorescent IRDye800CW (LI-COR Biosciences) was incubated with increasing concentrations of LYVE-1 and reacted for 30 minutes with the cross-linker BS3. Bovine serum albumin (BSA) and unlabeled FGF2 were used as negative controls. Cross-linking samples were analyzed by 10% SDS-PAGE under reducing conditions. The IR signal was visualized using Odyssey infrared imaging system (LI-COR Biosciences). The arrow shows cross-linked FGF2/LYVE-1 oligomers. (C) Co-immunoprecipitation of FGF2/LYVE-1 complexes in solution. Premixed FGF2-N-His/LYVE-1 (500/150nM) was incubated either with mix of 2.6 μg anti–LYVE-1 antibody and 50 μL Dynabeads protein G beads and revealed with rabbit anti-FGF2 antibody and secondary donkey anti–rabbit IRDye800CW conjugated antibody (lane 1) or with mix of 3.3 μg anti-FGF2 antibody and 50 μL Dynabeads protein G beads and revealed with mouse anti–LYVE-1 antibody and secondary donkey anti–mouse IRDye800CW conjugated antibody (lane 2). M, protein molecular weight markers.