Correlation between total ferritin (ferritin), IRP, and LIP levels in K562 cells after acute iron depletion. K562 cells incubated for 30 minutes without additives (Con) or with 100 μmol/L SIH (ISAH) were allowed to “recover” for 8 hours at 37°C in bicarbonate-free DMEM containing the indicated additives: 0.1% dimethyl sulfoxide (DMSO) final (Con-DMSO, ISAH-DMSO), 20 μg/mL leupeptin (Con-Leup, ISAH-leup), or 10% FCS (ISAH-FCS). ‘Untreated’ cells (left bar) were grown in full medium without additives for 8 hours. After the 8-hour period, aliquots of cells were taken for determination of LIP levels by the calcein method as in Fig 1(lower panel, ▪), ferritin by ELISA (lower panel, □), and IRP by electromobility gel retardation assay (upper panel). LIP levels are given in micromoles per liter, ferritin values are expressed relative to control (which contained ∼130 ng/mg protein), and IRP is given in terms of densitometry tracings (OD) of gel shift bands relative to control. All samples in the gel were obtained from equivalent numbers of cells. Rec-IRP refers to recombinant IRP, which is used here as standard for identification purposes. The second row of the upper panel depicts gel shifts performed after treatment with β-mercaptoethanol (βME) representing total (fully activated) cellular IRP.