Effect of NO on IRP-1 and IRP-2 activity of macrophage J774 cells. (A) J774 cells were left untreated or treated with the NO donor SNAP (0.5 mmol/L) for 24 hours as indicated. IRP activity in cytoplasmic extracts was determined by bandshift assay using the pSPT-fer probe as described in the legend to Fig 1. The results shown are representative of seven separate experiments. (B) The mutant CG284 probe, which is specific for IRP-2, was incubated with lysates (10 μg protein) of J774 cells untreated and treated with 0.5 mmol/L SNAP for 24 hours as indicated. The pSPT-fer probe was incubated with lysates (2 μg protein) of untreated cells. Formation of RNA/protein complexes was estimated by bandshift assay as described in the legend to Fig 1. The autoradiogram shown is representative of four separate experiments.