HFE increases ALK3 protein expression in vitro and in vivo. (A) HFE increases ALK3 protein expression in Hep3B cells. Cells were transfected with and without a constant amount of ALK3-HA plasmid in the presence of increasing amounts of HFE-Myc. Forty-eight hours after transfection, cells were harvested for western blotting for ALK3 and HFE protein levels. (B) HFE does not alter ALK3 mRNA expression. Hep3B cells were transfected with and without ALK3-HA in the presence or absence of HFE-Myc. Forty-eight hours after transfection, cells were harvested for real-time PCR analysis for ALK3 mRNA levels. (C) HFE increases endogenous ALK3 protein expression in Hep3B cells. Cells were transfected with or without ALK3-HA in the presence or absence of HFE-HA plasmid. Forty-eight hours after transfection, cells were harvested for western blotting for endogenous ALK3 and transfected ALK3-HA protein using an ALK3 antibody from Millipore (left panel). The bands were quantified by densitometry (right panel). (D) ALK3 protein expression in WT and Hfe knockout (KO) mice. Liver lysates from WT and Hfe KO mice at 8 to 10 weeks of age were used for western blotting for ALK3 protein. Livers from Alk3^fl/fl; Alb-Cre and the littermates Alk3^fl/fl were included to determine the specific ALK3 protein bands (n = 3). The bands were quantified by densitometry (bottom panel). (E) Livers from WT and Hfe KO mice were analyzed for ALK3 mRNA expression. Rpl19 or RPL19 are the internal controls for real-time PCR. *P < .05, **P < .01. Data are represented as mean ± SD (n = 3 for panels B-D; n = 4 for panel E). All experiments were repeated 2 to 3 times.