HoloTf increases HepG2 cell TfR2 protein but not mRNA. HepG2 cells were cultured 24 hours with or without the indicated additions of holoTf, apoTf, or non–Tf-bound iron (NTBI), and cell lysates were prepared for Western blot analysis as detailed in “Materials and methods.” (A) HepG2 cells were treated with indicated concentrations of holoTf, and lysates (120 μg protein) were electrophoresed on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted to detect TfR2 protein levels. (B) HepG2 cells were incubated with or without 30 μM holoTf for the indicated times, and TfR2 levels were also determined by Western analysis. (C) HepG2 cells were treated with or without 30 μM holoTf, 30 μM apoTf, 65 μM Fe-NTA, or 100 μM Fe-NTA, and immunoreactivity was determined for TfR2, TfR1, and ferritin protein levels. To control for loading, tubulin levels were also determined. Representative results from one of at least 2 experiments are shown. (D) Total RNA samples were isolated from HepG2 cells incubated with 30 μM holoTf for 24 hours. RNA (56 μg) was electrophoresed on a 0.9% agarose gel and transferred to Nytran N membrane for Northern blotting. The Northern blot was hybridized with ³²P-labeled TfR2 probe, then stripped and reprobed for TfR1 and β-actin. Hybridized probes were detected by phosphorimaging (Quantity One software; Bio-Rad).