Wild-type Nramp2 colocalizes with cell-associated transferrin and is expressed on the cell surface. Expression constructs containing epitope-tagged Nramp2 cDNAs were transfected into HEK293T cells. (A) Two days posttransfection, cells were allowed to take up Texas-red transferrin, and then permeabilized for FITC immunostaining of the FLAG-Nramp2 proteins. Cells transfected withNramp2 in the antisense (-) orientation (upper left panel) did not stain for FLAG and served as a negative control. These cells did, however, take up Texas-red transferrin as expected (upper right panel). Cells transfected with FLAG-Nramp2 in the sense orientation show colocalization of Nramp2 protein with transferrin (bottom panels). Arrows highlight similarities in punctate staining of intracellular structures. (B) Two days posttransfection, cells were incubated with a membrane-impermeable form of biotin to label cell surface proteins. Cells were then solubilized in RIPA buffer, and FLAG-Nramp2 was immunoprecipitated using a specific antibody against the FLAG epitope. Proteins were resolved on a 10% SDS-polyacrylamide gel, transferred, and probed with streptavidin-HRP to detect biotin using enhanced chemiluminescence. Cells transfected with FLAG-Nramp2 in the sense direction resulted in abundant immunoprecipitation product (lane 2, arrow). Cells transfected with Nramp2 in the antisense direction did not produce any immunoprecipitation product (lane 3). Sizes of molecular weight standards (lane 1) are indicated on the left. In this system, we consistently find that the apparent molecular weight of Nramp2 is smaller than the predicted molecular weight of 63 kD.