Experimental definition of IRP1-binding sites using EMSA. (A) Schematic representation of an IRE motif. Squared region indicates the IRE core region predicted by SIREs software. (B) Schematic representation of the H-ferritin wild-type and mutant IRE used for electrophoretic mobility shift assays. Deletion of C14 in the IRE is indicated by a red cross. (C) Competitive EMSA analyses with 24 different H-ferritin IRE variants (mutants A to Y). Values and standard errors for competition with the H-ferritin wild-type and the δ-C mutant IRE are highlighted in gray. In the table, mutated positions are indicated according to the nomenclature shown in panel A, and mutated nucleotides are underlined. Values for fold difference between the different variants versus the H-ferritin IRE wild-type (IRE wt) at 40× fold molar excess are reported. P values comparing each variant with the H-ferritin IRE mutant, (IRE mut) are reported (***P < .001, **P < .01, *P < .05; unpaired 2-tailed Student t test). Mutants that do not reach statistical significance are filled in red. The red dashed line indicates the value for Aco2 5′IRE. Graph shows percentage of raw signal for competitive EMSAs at 40× molar excess of the indicated competitor. Data are presented as mean ± SEM from a minimum of 3 experiments.