Figure 4.

The first external loop region of mZIP4 is required for high-affinity zinc-stimulated endocytosis. A–C, HEK293 cells stably expressing WT and mutant mZIP4 proteins were preincubated with Chelex-treated medium for 3 h to increase surface levels of the proteins. Cells were then allowed to internalize anti-HA antibodies from Chelex-treated medium or the same medium containing the indicated zinc concentrations for 5 min. Immunoblots were then used to detect levels of internalized anti-HA antibodies endocytosed by WT and mutant mZIP4-HA proteins. Tubulin levels shown in the lower panel demonstrate equal protein loading. D, quantification of internalized anti-HA antibodies in HEK293 cells expressing WT and mutant mZIP4 proteins. Data are shown as relative fold-changes compared with basal condition of WT mZIP4-HA (mean ± S.D.) from multiple replicates per condition. Statistics: two-way ANOVA, Dunnett's post hoc test (*, p < 0.05).