FIG. 7.

The processing and degradation of ZIP4 are regulated by zinc and require endocytosis. (A) Hepa cells cultured in medium containing 10% Chelex-treated FBS (CX) for 24 h were treated with the indicated concentrations of ZnSO₄ (4, 20, or 100 μM) for the indicated times (0.5 to 6 h). Membrane proteins (20 μg) were analyzed by Western blotting using an anti-peptide ZIP4 antibody (top) or a ZIP1 antibody (bottom). Lane N, cells cultured in medium containing normal FBS. (B) Hepa cells were cultured in zinc-deficient medium as in panel A, treated with the indicated inhibitors for 30 min or not treated (−) and then incubated with 4 μM ZnSO₄ for 2 h in the presence of inhibitor. Western blotting of membrane proteins (20 μg) was performed using an anti-peptide ZIP4 antibody (top) or a ZIP1 antibody (bottom). Endocytosis inhibitors used were as follows: Mβ, 10 mM methyl-β-cyclodextrin; Suc, 350 mM sucrose.