HFE expression stimulates IRP activity. (A) Cytoplasmic extracts of HtTA or HtTA-HFE cells grown with or without doxycycline (1 μg/mL for 12 days) were prepared and subjected to gel retardation assays. Where indicated (lanes 5 to 9), 2-mercaptoethanol (2%) was added to the sample (25 μg of protein/lane) before the RNA probe. Positions of IRE/IRP complexes and unbound RNA (free probe) are indicated by arrows. (B) Gel retardation assays as above, except that doxycycline treatment of the cells was performed for the times indicated. For each time point, the amount of IRP/IRE complexes formed in the absence of doxycyline was set at 100%; the percentages of IRP/IRE complexes in the presence of doxycycline are given below the lanes. Quantitative determination of IRP/IRE complexes was done by phosphorimaging.