Time course of IRP activity in cytokine-treated macrophage J774 cells. J774 cells were left untreated (lane 1) or treated for 4 (lane 2) or 24 hours (lanes 3,4) with cytokines (100 U/mL IFN-γ plus 1 μg/mL LPS); where indicated, the iNOS inhibitor NMMA (0.1 mmol/L) was also present (lane 4). A total of 2 μg protein of cytoplasmic extracts was analyzed for IRE-binding activity by RNA gel retardation assay with excess ³²P-labeled probe containing IRE sequences in the absence (upper panel) or presence (lower panel) of 2% 2-mercaptoethanol. TNF-α and nitrite production was assayed in the culture medium as described in the legend to Fig 1.