Regulation of IRE-binding activity of IRPs by IFN-γ/LPS in J774 macrophages. (A) Cells were treated overnight in RPMI culture medium containing 10% FCS with 100 μmol/L DFO (D), 5 to 500 U/mL IFN-γ (I), 10 to 1,000 ng/mL LPS (L), and/or 500 μmol/L NMMA (N), or remained untreatred (control, C). Twenty micrograms of detergent cell extract was assayed for IRE-binding activity of IRP1 and IRP2 by gel-shift assay in the absence or presence of 2% 2-ME. Nitrite production was assayed in the culture medium by the Griess reaction. Results shown are representative of 4 separate experiments. (B) Autoradiographs were densitometrically scanned and the proportion of spontaneous IRP1 activity was expressed as a percentage of the value obtained after exposure to 2% 2-ME, which allows calculation of total IRE binding activity of IRP1. IRP2 activity was plotted in arbitrary units.