Detection of the Nramp1 and Nramp2 cMyc-tagged proteins in CHO cell membranes. (A) Schematic representation of the two mRNAs corresponding to the IRE-containing form and to the non-IRE form ofNramp2 generated by alternative splicing of a 3′ end exon (see Lee et al40). The two mRNAs harbor distinct 3′ untranslated region (3′UTR) and encode two polypeptides with distinct carboxy termini (hatched boxes). The mN2 (NT) antibody was raised against an amino terminal segment of Nramp2 that is common to the two Nramp2 protein isoforms (solid boxes). The mN2 (CT) antibody was raised against the carboxy terminal extremity of the protein encoded by the Nramp2 non-IRE mRNA (Nramp2 isoform II). (B) Crude membrane extracts from untransfected CHO cells (lanes 1, 3, 5, 7, 9, 11, 13, and 15), from Nramp1 transfectants (lanes 10, 12, 14, and 16), and from Nramp2 isoform II transfectants (lanes 2, 4, 6, and 8) were separated by SDS-PAGE on a 10% acrylamide gel followed by transfer to PVDF membranes. Immunodetection was with affinity-purified mNramp1 [mN1 (NT); lanes 1, 2, 9, and 10], mN2 (NT; lanes 3, 4, 11, and 12), and mN2 (CT; lanes 5, 6, 13, and 14) antibodies and monoclonal anti-cMyc antibody 9E10 (lanes 7, 8, 15, and 16), as described in Materials and Methods. The positions and sizes (in kilodaltons) of molecular weight markers are shown.