HFE interacts with ALK3 and synergizes with ALK3 to stimulate hepcidin expression. (A) COS7 cells were transfected with HFE-Myc in the presence or absence of ALK3-HA. Cell lysates were used for immunoprecipitation (IP) and subsequent western blot analysis (WB) using indicated antibodies. Cell lysates were used for WB as indicated to show inputs. (B) COS7 cells were transfected with ALK3-HA in the presence and absence of HFE-Myc. Cell lysates were used for IP and /or WB as indicated. (C) HepG2 cells were transfected with HFE-Myc in the presence or absence of ALK3-HA. Cell lysates were used for IP and /or WB as indicated. (D) Hep3B cells were transfected with ALK3-Flag in the presence of empty vector, HFE-HA (WT), HFE-HA (C282Y), or HFE-HA (H63D). Cell lysates were used for IP and /or WB as indicated. (E) Hep3B cells were transfected with or without HFE-Myc. Cell lysates were used for IP and /or WB as indicated. (F) Lysates from WT and Hfe^∆CD-Myc transgenic mice were used for IP and/or WB as indicated. (G) Hep3B cells were transfected with ACTRIIA-Myc in the presence of empty vector, ALK3-HA, or HFE-HA. Cell lysates were used for IP and /or WB as indicated. (H) Hep3B cells were transfected with empty vector (0), ALK3-HA alone, HFE-Myc (20 ng/mL) alone, or ALK3-HA in combination with HFE-Myc expression plasmids. Forty-eight hours after transfection, cells were harvested for real-time PCR analysis for hepcidin and RPL19 mRNA levels. RPL19 is the internal control. *P < .05, **P < .01, ***P < .001. Data are represented as mean ± SD (n = 3 for panel F). All experiments were repeated 2 to 3 times.