HEK293 cell transfected with expression constructs encoding SLC39A5 wild-type (WT), Y47*, R311*, and R322* variants. (A, B) Immunostaining and FACS analysis demonstrating WT SLC39A5 localization to the cell surface. Scale bar, 10 um. (C) Overexpression of WT SLC39A5 results in Zn2 + mediated MRE activation in a dose-dependent manner, n=4. (D) FACS analyses demonstrate that cell surface expression of SLC39A5 Y47*, R311*, and R322* muteins is markedly reduced. (E) Variants Y47*, R311*, and R322* did not mediate Zn2 + induction of MRE, n=8, Statistical comparison to Mock and WT, respectively: ***p<0.001, ###p<0.001, two-way ANOVA with post hoc Tukey’s test. Metal regulatory element (MRE), firefly luciferase (Fluc), renilla luciferase (Rluc), cytomegalovirus (CMV), gene of interest (GOI), internal ribosome entry site (IRES).