Neutralizing anti-ERFE antibodies bind to the N-terminal region of ERFE. (A) Anti-ERFE antibodies (15.1, 20.1, and 28.1) were assayed at different concentrations (10⁻⁵-10² nM) in ELISA plates coated with full-length ERFE, F4 (N-terminal domain region), and the globular C1q domain. (B) HTRF assay for detection of binding competition using cryptate-labeled anti-ERFE antibodies (20.1 and 28.1) and BMP6 (0.1-200 nM) and unlabeled antibody as positive control. 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. (C) Huh7 cells were treated for 24 hours with 200 ng/mL of murine ERFE alone or in combination with 10 µg/mL of anti-ERFE antibodies 15.1 and 20.1, and analyzed for HAMP gene expression. Bars represent mean ± standard deviation. n = 3 replicates per group. ***P < .001; ****P < .0001 using the Student t test.