Subcellular distribution of WT and Nramp2^G185R. LLC-PK₁ cells stably transfected with either WT (A, C, E) or G185R (G, I, K) Nramp2-HA were immunostained with anti-HA antibodies following fixation and permeabilization, to reveal the total cellular Nramp2. The corresponding panels, B, D, F and H, J, L, were stained as follows: B and H, surface Nramp2 was stained selectively by treatment with anti-HA Ab prior to permeabilization, as described in “Materials and methods”; D and J, the cells were incubated with rhodamine-conjugated transferrin for 2 hours prior to fixation to label early endosomes; F and L, permeabilized cells were immunostained with anti-calnexin antibodies. Insets in E and F are magnifications of the indicated areas. Images are representative of at least 3 independent experiments of each type. Scale bars equal 5 μm.