mRNA-binding activity of IRP is independent of iron in differentiating primary erythroblasts. (A) Determination of apparent (left panels) and total IRP mRNA-binding activities (right panels; +2-ME43 ) in extracts of mouse erythroblasts (designated “FL,” for fetal liver–derived cells), pretreated as described in Figures 2 and 3. Electrophoretic mobility shift assays (EMSAs) of complexes between IRP and radiolabeled in vitro–transcribed RNAs containing the IREs of mouse FerH mRNA (clone 42)42 were performed as described in “Materials and methods.” Control extracts, demonstrating the full regulatory potential of IRP, were prepared from mouse embryo fibroblasts (MEFs). Total IRP1 and IRP2 protein (B) and mRNA levels (C) were determined by Western (Erk1/2 used as loading control) and Northern blotting (28S rRNA signal as RNA quality and loading control), respectively.
