Regulation of TfR2 occurs at the protein level. (A) TfR2 transcript in HepG2 cells does not increase in response to diferric Tf. Total RNA was isolated from approximately 1 × 10⁷ HepG2 cells 24 hours after the addition of 25 μM diferric Tf or an equal volume of HBS to the medium. Expression of TfR2, TfR1, and GAPDH transcripts was measured by real-time qRT-PCR analysis of cDNA synthesized from 2 μg total RNA. Levels of TfR2 and TfR1 transcripts are shown relative to GAPDH levels. The graph represents the mean of 3 separate experiments in which each sample was analyzed twice in triplicate. Error bars depict SD. (B) Diferric Tf stabilizes TfR2 protein. HepG2 cells seeded at 2 × 10⁴ cells/cm² were incubated in normal medium or medium with 25 μM diferric Tf for 24 hours before the addition of 100 μg/mL cycloheximide for 4, 3, 2, 1, 0.5, and 0 hours. Cells were solubilized, lysates from duplicate wells were pooled, and half of each sample was analyzed by Western blot. TfR2 and TfR1 were detected with fluorescence-labeled secondary antibodies for quantification and then with HRP-conjugated secondary antibodies for chemiluminescence imaging. The integrated intensity of TfR2 was normalized to that of TfR1, which did not change detectably over the time-course of the experiment. The normalized intensity was expressed as a percentage of the normalized intensity at time 0, and the log of this value was plotted. Half-life was determined by linear regression analysis. The graph shows the mean of 3 experiments. Error bars indicate average deviation from the mean.