Iron-dependent modulation of IRP-binding activity to the ferH-IRE in primary versus transformed ebls. (A) IRP-IRE-binding activity was determined in vitro by a gel retardation assay as outlined in Materials and Methods. Aliquots of the cell extracts from self-renewing ebls used for immunoblotting (see Fig 3) were incubated with an excess of radiolabeled ferH-IRE transcripts and the resulting RNA-protein complexes were separated in nondenaturing polyacrylamide gels. Where indicated, maximal IRP1-IRE binding activity was estimated by treatment of the extracts with 2-mercaptoethanol (2-ME) in vitro. The amount of radioactivity in the IRP-IRE complexes was quantified by phosphoimaging. Numbers below the pair of corresponding lanes represent the fold induction of IRP activity. (B) Levels of IRP1 (left panel) and IRP2 (right panel) in transformed versus primary ebls cultivated under different iron supply, as detected by immunoblotting. (C) IRP1 versus IRP2 binding activity to the ferH-IRE in extracts of transformed and primary ebls treated for 24 hours with Des. Gel retardation assays in combination with antibody-mediated supershifts were performed as described in detail under Materials and Methods. As a control (no extract), lysis buffer was incubated with 3 μL antibody and the radioactive ferH-IRE probe. -IRP1 and -IRP2, IRP1 and IRP2 specific antisera, respectively; IgG, nonspecific antibody.