ERFE suppresses BMP/SMAD signaling by inhibiting BMP5, BMP6, and BMP7. (A) Venn diagram representing the number of genes differentially expressed by Huh7 cells treated with murine Erfe (10 µg/mL) compared with vehicle-treated cells and analyzed by Illumina microarray or RNA sequencing (RNA-seq). (B) Gene expression measured by qRT-PCR of the common 4 differentially expressed genes in Huh7 cells treated with vehicle or murine Erfe (10 µg/mL). (C) Huh7 cells treated with mouse ERFE (10 µg/mL), BMP6 (6 nM or 18 nM), and LDN (100 nM), alone or in combination, for 30 minutes. pSMAD1/5/8 and SMAD1 were analyzed by western blot. pSMAD:SMAD ratios were calculated by densitometry. (D) C2C12 BRE-Luc cells were treated with 2 nM BMP in combination with a gradient of mouse ERFE concentrations (7.5 pM to 0.5 µM) for 24 hours, and luminescence was measured in each well. Data were normalized to percentage of maximum luminescence (no ERFE). (E) Huh7 cells treated with 2 nM BMPs alone or in combination with 10 µg/mL of mouse ERFE in serum-free media and analyzed 6 hours after treatment for HAMP gene expression by qRT-PCR. (F) Homogeneous time-resolved fluorescence assay for detection of binding between ERFE and BMP, using cryptate-labeled anti-ERFE antibody and BMPs (0.1-200 nM) and unlabeled antibody as positive control. Values were calculated as %ΔF = [(F665 sample/F615 sample) − (F665 control/F615 control) (F665 control/F615 control)] × 100, in which control is the background fluorescence energy transfer in wells containing labeled antibody alone. Results for panels B-F represent mean ± standard deviation from 3 independent experiments. *P < .05; **P < .01; ***P < .001; ****P < .0001 by Student t test. RLU, relative light units.