Subcellular localization of ZIP1 and ZIP3 in RWPE1 and RWPE2. a. Localization of ZIP1 and ZIP3. RWPE1 and RWPE2 were seeded at ~50% confluence in a 4-well slide chamber and cultured at 37°C for 48 hours. Cells were then treated with 0 or 50 μM ZnSO4 at 37°C for 2 hours before immunofluorescent staining. The endogenous ZIP1 and ZIP3 proteins in RWPE1 and RWPE2 were detected by a chicken anti-ZIP1 antibody and a rabbit anti-ZIP3 antibody, respectively. An Alexa 488-conjugated goat anti-chicken (ZIP1) or anti-rabbit (ZIP3) antibody were used as secondary antibodies for the photomicrographs. Arrows indicate the plasma membrane localization of ZIP1. b. Localization of ZIP3 and LAMP1 in RWPE2. RWPE2 were seeded at ~50% confluence in a 4-well slide chamber and cultured at 37°C for 48 hours. Cells were fixed, permeablized, and stained as described in the Methods. The green (ZIP3, panel A) and red (LAMP1, panel B) florescent images were merged to indicate the area of overlap (panel C), which is shown in yellow.