Gel-retardation assay.
(A) Human IRP1 and PfIRPa bind a mammalian IRE in a concentration-dependent manner. Gel-retardation assays of IRP1- and PfIRPa-mammalian IRE complexes are shown. The conditions are as described in the legend to Figure 2. Increasing amounts of the32P-IRE were incubated with 6 μg protein from P falciparum and the human EBV cell line expressing IRP1. Lane 1, IRE alone; 2-4, PfIRPa-IRE; 5-7, uninfected RBC lysate-IRE; 8-10, IRP1-IRE. Lanes 2, 5, and 8, 0.125 pmol 32P-IRE per lane; lanes 3, 6, and 9, 0.5 pmol 32P-IRE per lane; lanes 4, 7, and 10, 2 pmol 32P-IRE per lane. (B) Cold IRE competes with32P-IRE for the binding to PfIRPa. Lanes 1 and 2, free32P-IRE; 3 and 4, 10 μg lysate protein + 1 pmol32P-IRE; 5, 10 μg lysate protein + (1 pmol32P-IRE + 25 pmol cold IRE); 6, 10 μg lysate protein + (1 pmol 32P-IRE + 50 pmol cold IRE); 7, 10 μg lysate protein +(1 pmol 32P-IRE + 75 pmol cold IRE).
