Analysis of FGF2/LYVE-1 interaction by AlphaScreen-based technology. (A) Detection of FGF2/LYVE-1 interaction. Top panel: Assay design of the AlphaScreen experiment. GST tagged FGF2 (FGF2-N-GST) was bound to AlphaScreen glutathione donor beads and 6xHis-tagged LYVE-1 (LYVE-1 N-6xHis) to AlphaScreen Ni chelate acceptor beads. Bottom panel: Direct interaction between FGF2 and LYVE-1. The recombinant proteins of indicated concentrations were incubated with donor and acceptor beads for 1 hour at room temperature before signal measurement. (B-C) AlphaScreen-based competition assays. The interaction between 500nM FGF2-N-GST and 50nM LYVE-1 N-6xHis was competed in the presence of increasing concentrations of heparin (B) and PDGF-BB (C). IC50 were determined with equation 1-site competition (GraphPad Prism Version 5). (A-C) Results are representative experiments from 3 independent experiments. Results are mean values ± SD (n = 3).