ERFE binds to BMP5 and inhibits BMP5 signaling to hepcidin. (A) BMP5 (2 μg) or BMP6 (1 μg) was mixed with 1 μL of Flag-ERFE concentrate (52.9 ng/μL) supplemented with a protease inhibitor cocktail. After saving 3% of the total mixture as input, the solutions were incubated with 10 μL of Flag M2 agarose at 4°C overnight. The immunoprecipitated complexes were washed and eluted with 150 μg/mL of 3× Flag peptide in tris-buffered saline for 30 minutes at 4°C. The inputs and eluents were immunoblotted using antibodies against BMP5, BMP6, and Flag. The experiments were repeated 3 times, with 1 representative blot shown. (B) Hep3B cells were transfected with Flag-ERFE or an empty vector (EV). After 48 hours, cells were treated with 5 ng/μL BMP5 for 6 hours. HAMP relative to RPL19 mRNA was analyzed via qRT-PCR. (C-F) Eight-week-old male (open circles) and female (closed circles) single Bmp5^se and littermate control Bmp5^wt mice (C,E) or double Bmp5^se-Bmp6^fl-Tek-Cre⁺ and littermate control Bmp5^se-Bmp6^fl-Tek-Cre^– mice (D,F) were given 200 units of Epogen (EPO) or phosphate buffered saline (PBS) intraperitoneally. After 15 hours, bone marrow Erfe (C,D) and liver Hamp (E,F) relative to Rpl19 mRNA were analyzed via qRT-PCR. The average of male Bmp5^wt or Bmp5^se-Bmp6^fl-Tek-Cre^– control mice treated with PBS was set to 1. For panel B, bars represent the mean ± SEM from 4 independent experiments. ∗∗∗P < .001 for the indicated comparisons by 2-way ANOVA with Tukey post hoc test. For panels C-F, individual data points are shown, and bars represent the mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 for EPO relative to PBS treatment for sex- and genotype-matched mice using Student t test.