Identification of the Pfn2 3′ UTR IRE and IRP binding studies. (A) Schematic representation of the Pfn2 mRNA. Coding region (black box), Pfn2 XcmI fragment, and fragments A to E and IRE are shown. (B). Human and mouse 3′ IRE motifs in Pfn2, Tfrc, and Slc11a2 mRNAs. Notice differences in sequence in the apical loop. The arrow indicates the deletion of the adenosine (ΔA, mut) used as mutant construct. (C) Nonradioactive competitive EMSA using fluorescent ferritin H IRE-labeled probe and twofold molar excess of unlabeled competitors (full-length Pfn2 mRNA or Pfn2 mRNA fragments). Constructs or fragments (XcmI fragment, A to E fragment) used as competitors are indicated. Mut, fragment A containing a single deletion in the first A of the 6-nucleotides apical loop; WT, wild-type fragment A. One representative experiment out of 4 is shown. (D) Nonradioactive competitive EMSA using fluorescent ferritin H IRE-labeled probe and 80-fold molar excess of unlabeled competitors (ferritin H or mouse and human Pfn2 IRE sequences). (E) Representative phylogenic conservation of the Pfn2 IRE among mammals; an extended alignment is shown in supplemental Figure 2. Asterisks (*) indicate residues conserved in all aligned species. CDS, coding sequence.