HFE lowers ferritin levels in TRVb cells. (A) HFE expression. TRVb or TRVb/HFE/β2M cells were left untreated (C) or incubated with 3 mg/mL diferric Tf or 150 mM Fe-NTA for 24 hours prior to harvesting. Cell lysates (30 μg total protein) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to HFE (no. 137), actin, 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. TRVb cells were treated with 0.5 μM sHFE overnight in 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 in panel A. This experiment was repeated 3 times using 3 different clones for each cell line with similar results.