HFE decreases intracellular ferritin levels in TRVb1 cells. (A) HFE expression. TRVb1 or TRVb1/HFE/β2M cells were left untreated or incubated with 3 mg/mL diferric Tf or 150 μM Fe-NTA for 24 hours prior to harvesting. Cell lysates (45 μg total protein) were subjected to SDS–polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose, and probed with antibodies to TfR1, actin, HFE (no. 137), ferritin (Ft), or β2M. The no. 137 rabbit anti-HFE serum gives a nonspecific band just above the HFE doublet. Actin was used as a loading control. (B) sHFE treatment. TRVb1 cells were treated with 0.5 μM sHFE overnight in the presence (Fe-NTA) or absence (C) of 150 μM Fe-NTA. Cell lysates were then prepared and used for immunodetection of ferritin levels as described for panel A. These experiments were repeated 3 times using 3 different clones for each cell line with similar results.