Expression of WT and Nramp2^G185R in stably transfected cells. (A) Lysates of LLC-PK₁ cells (left) or CHO cells (right) stably transfected with either the WT or G185R forms of HA epitope–tagged Nramp2 were subjected to electrophoresis and immunoblotting with anti-HA antibodies. Two separate clones of LLC-PK₁ cells transfected with mutant Nramp2 are illustrated. The positions of the complex-glycosylated (▦) and core-glycosylated forms of Nramp2 (□) are indicated to the left and the locations of molecular weight markers are shown to the right. Immunoblot is representative of 4 experiments. (B) LLC-PK₁ cells stably transfected with either the WT or G185R forms of Nramp2 were either left untreated (control) or were incubated with 2.5 μg/mL tunicamycin for 16 hours before solubilization, electrophoresis, and immunoblotting as for panel A. Representative of 4 experiments. (C) Quantification of the relative proportion of complex-glycosylated and core-glycosylated Nramp2 in LLC-PK₁ cells transfected with WT or mutant Nramp2. Immunoblots from experiments like that in panel A were scanned and the optical density of the complex- and core-glycosylated forms were then expressed as a percentage of the total. Data are means plus or minus SE of 8 experiments.