Figure 4. ZIP13G64D protein is degraded by a ubiquitination-dependent pathway.
A Treatment with PYR-41, a ubiquitin E1 inhibitor, suppressed the downregulation of ZIP13G64D protein in the presence of cycloheximide (CHX). HeLa cells stably expressing WT-V5 or G64D-V5 were treated with 10 μM MG132 or 10 μM PYR-41 together with CHX for the indicated times. Total cell lysates were subjected to Western blotting analysis with an anti-V5 antibody. Right panel shows the relative expression levels of ZIP13 proteins. Data are representative of two independent experiments.
B HeLa cells stably expressing WT-V5 or G64D-V5 (Supplementary Fig S2A) were treated with 10 μM MG132 for 6 h. The cell lysates were analyzed by Western blot using an anti-V5 antibody. The ubiquitinated/non-ubiquitinated G64D protein ratio was upregulated compared to that of wild type (right panel). Data are shown as mean ± s.e.m. (*P = 0.036).
C Single cycle kinetic analysis of ZIP13 protein binding to the amine-coupled antibody 35B11 on a Biacore sensor tip. Solution-phase ZIP13-35B11 binding was measured by surface plasmon resonance (BIAcore). A representative BIAcore sensorgram shows the response over time (resonance units [RU]) during the binding of purified recombinant human ZIP13 protein to immobilized 35B11 antibodies. Purified human ZIP13 protein at concentrations of 25, 50, 100, 200, and 400 nM was added at 0, 190, 380, 570, and 760 s, respectively. The graph is representative of four independent experiments.
D Intracellular flow cytometric analysis of the endogenous ZIP13 expression in a healthy female donor or female SCD-EDS patient. Cultured primary human fibroblasts were treated with DMSO or 10 μM MG132 for 6 h. After fixation and permeabilization, the cells were stained with the monoclonal antibody 35B11, followed by goat anti-mouse Alexa 488. Data are representative of two independent experiments. Similar results were obtained in a healthy male donor and male SCD-EDS patient.
Source data are available online for this figure.