Regulation of IRP activity by extracellular ferritin. Cells were incubated with medium only (control, lane 2), 100 μmol/L ferric ammonium citrate (FAC, lane 3), 40 nmol/L H-rich holoferritin or apoferritin (lanes 4 and 5, respectively), or with 50 μmol/L DFO (lane 6) for 64 hours, beginning at day 6 of phase II. Fresh cytoplasm extracts (10 μg protein) were analyzed for IRP activity by electromobility-RNA gel retardation. In lane 1, a 100-fold excess of unlabeled IRE was added as a specific competitor to the labeled IRE. Cell lysates were analyzed without β-mercaptoethanol (2-ME) (top panel) or after incubation with 2% 2-ME for 5 minutes at room temperature (bottom panel).