Modulation of IRP-1 and IRP-2 activity in cytokine-treated macrophage J774 cells. J774 cells were left untreated or treated for 24 hours with cytokines (100 U/mL IFN-γ plus 1 μg/mL LPS); where indicated, the iron chelator DFO (0.05 mmol/L) was also added. Iron loading was obtained by incubating cells with 50 μg/mL ferric ammonium citrate for 24 hours (lane 3). A total of 2 μg protein of cytoplasmic extracts was analyzed for IRE-binding activity by RNA gel retardation assay with excess ³²P-labeled RNA transcribed from the pSPT-fer probe containing the IRE of the ferritin H mRNA in the absence (upper panels) or presence (lower panels) of 2% 2-mercaptoethanol. TNF-α and nitrite production was assayed in the culture medium as described in Materials and Methods. TNF-α units were calculated from a standard curve with rTNF-α used as internal standard for each test.