Expression of HFE in TRVb cells decreases Fe⁺² uptake. Cells (∼ 1 × 10⁶ in 35-mm dishes) were incubated with 1 mL incubation buffer (25 mM Tris, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 25 mM 2-N-morpholino ethane sulfonic acid, 1.8 mM CaCl₂, pH 5.5) containing 1 mM ascorbic acid and 10 μM ⁵⁵Fe⁺² for 30 minutes at 37°C, washed with ice-cold PBS plus 5 mM EDTA, and lysed in 0.1 M NaOH. ⁵⁵Fe uptake was determined by liquid scintillation counting of the lysates. Student t test demonstrated that ferrous iron uptake in the TRVb cells was significantly higher than in cells expressing TRVb/HFE/β2M (^*P < .01). The same finding was true in TRVb1 cells versus TRVb1/HFE/β2M. This experiment was repeated 4 times using 3 different clones for each cell line with similar results. Error bars indicate standard deviation (SD).