HFE stabilizes ALK3 protein by inhibiting its ubiquitination and proteasomal degradation. (A-B) HFE inhibits ALK3 degradation. Hep3B cells were transfected with ALK3-HA in the presence or absence of HFE-Myc. Forty-six hours later, cells were incubated with Chx for 0, 1, 2, and 4 hours. Cells were then harvested and subjected to western blot analysis for ALK3 protein levels using anti-HA antibody. A representative western blot is shown in (A). Densitometric analysis is shown in (B). (C) ALK3 degrades primarily through the proteasomal pathway. Hep3B cells were transfected with empty vector or ALK3-Flag (200 ng/mL). Forty-six hours after transfection, cells were treated with increasing doses of NH₄Cl (0, 1, and 10 mM) or MG132 (0, 5, and 10 μM) for 5 hours. Whole-cell lysates were prepared for western blot analysis with anti-Flag antibody for ALK3 protein expression. (D) HFE attenuates ubiquitin-mediated ALK3 degradation. Hep3B cells were transfected with ALK3-Flag alone, or ALK3-Flag in combination with HA-ubiquitin in the absence or presence of HFE-Myc. Forty-eight hours after transfection, cells were harvested for western blotting for ALK3 and HFE protein levels. Representative western blot is shown in the left panel, and quantification of 3 separate experiments is shown in the right panel. (E) HFE inhibits ALK3 ubiquitination. Hep3B cells were transfected with ALK3-Flag alone, or ALK3-Flag in combination with HA-ubiquitin in the presence of increasing amounts of HFE-Myc. Forty-six hours after transfection, cells were treated with MG132 (5 μM) for 5 hours. Whole-cell lysates were precipitated with anti-Flag antibody and analyzed by western blotting for ubiquitinated ALK3 using anti-HA antibody. Whole-cell lysates were also used for western blot analysis for AKL3 and HFE expression. (F) HFE-H63D does not inhibit ALK3 ubiquitination. Hep3B cells were transfected with ALK3-Flag alone, or ALK3-Flag in combination with Myc-ubiquitin in the presence of HFE-HA (WT), HFE-HA (C282Y), or HFE-HA (H63D). Forty-six hours after transfection, cells were treated with MG132 (5 μM) for 5 hours. Whole-cell lysates were precipitated with anti-Flag antibody and analyzed by western blotting for ubiquitinated ALK3 using anti-Myc antibody. Whole-cell lysates were also used for western blot analysis for AKL3 and HFE expression. β-actin is the loading control. *P < .05, **P < .01. Data are represented as mean ± SD (n = 3 for panels A-B). All the experiments were repeated 2 to 3 times.