Cell-surface biotinylation of CHO cells expressing Nramp1/2HA or Nramp1/2Myc proteins. Cell-surface biotinylation was used to assess plasma membrane expression of Nramp1 (N1) and Nramp2 (N2) proteins modified either by insertion of an HA epitope into the loop delineated by predicted TM7/8 (panel A) or by a c-Myc epitope at the C-terminus (panel B). Live cells were labeled with membrane impermeant Sulfo-NHS-LC-biotin (see “Materials and methods”). Total cell protein extracts were prepared, and biotinylated proteins were isolated by affinity capture with streptavidin-agarose beads (from 100 μg of cell extract). Captured biotinylated proteins (B, the entire eluate), and postcapture supernatant (S; 10% of remaining supernatant volume) from CHO controls and from Nramp1/2HA (panel A) or Nramp1/2Myc (panel B) transfectants were analyzed by SDS-PAGE and immunoblotted with the corresponding anti–HA (panel A) or anti–cMyc monoclonal antibodies (panel B). Panel C shows a direct comparison of biotinylated proteins from CHO control, Nramp1HA, and Nramp1Myc expressing CHO transfectants immunoblotted with affinity-purified rabbit anti–Nramp1NT antibodies that recognize both Nramp1HA and Nramp1Myc proteins. The size of the molecular mass markers is shown to the left of the immunoblots.