Tf competes with HFE for binding to TfR1. Cells were incubated in the presence or absence of 10 mg/mL (∼ 125 μM) Tf and subjected to flow cytometry analysis as described in “Materials and methods,” using antibodies to detect TfR1 and HFE in separate experiments. (A) Increase in cell-surface TfR1 but not HFE in TRVb32-8/HFE-GFP-LDLR/β2M cells incubated with Tf. TRVb32-8 cells express a form of TfR1 lacking the endocytic signal, so internalization of this mutant TfR1 is dependent on interaction with HFE-GFP-LDLR, in which the cytoplasmic domain of LDLR mediates constitutive endocytosis. (B) Increase in cell-surface HFE but not TfR1 in TRVb1/HFE-GFP/β2M cells incubated with Tf. (C) Concentration dependence of the change in cell surface TfR1 in TRVb32-8/HFE-GFP-LDLR/β2M cells incubated with Tf. Cells were incubated in complete medium containing 0, 0.001, 0.01, 0.1, 1, 3, or 10 mg/mL diferric Tf, and cell surface TfR1 was measured by flow cytometry (the values for 0.001 and 0.01 were nearly identical and appear as a single point in this particular experiment). These experiments were repeated 3 times using 3 different clones for each cell line with similar results.