ERFE binds to BMP2, BMP4, and BMP6 with different affinities. (A) Binding kinetics of ERFE to BMP2, BMP4, BMP6, and GDF15 assessed by surface plasmon resonance (Biacore). No binding was observed for GDF15. Full-length human ERFE was immobilized in a CM4 chip. BMPs/GDF15 were tested at different concentrations (3.1-50 nM) for binding to immobilized ERFE and assayed for up to 60 seconds (2 replicates per condition). RU, resonance units. Apparent K_d (dissociation constant) was calculated for binding of BMP2, BMP4, and BMP6 to ERFE (assuming 1:1 binding interaction). (B) Apparent K_d of ERFE to BMP2, BMP4, and BMP6. (C) HTRF assay for detection of binding competition between BMPs (0.1-400 nM) and an anti-ERFE antibody. Unlabeled anti-ERFE antibody (anti-ERFE) and a control IgG antibody were used as positive and negative controls, respectively. Values calculated as %ΔF = [(F665 Sample/F615 Sample) − (F665 Control/F615 Control)/(F665 Control/F615 Control)] × 100. Data plotted as “% Control” represent the background fluorescence energy transfer in wells containing each labeled antibody, in assay buffer, alone.