ZIP13G64D protein is readily degraded by a proteasome‐dependent mechanism Proteasome inhibitor treatments. 293T cells were transfected with WT‐V5 or G64D‐V5.

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ZIP13G64D protein is readily degraded by a proteasome‐dependent mechanism Proteasome inhibitor treatments. 293T cells were transfected with WT‐V5 or G64D‐V5 ZIP13 and treated with 10 μM MG132 or 1 μM bafilomycin for 6 h. ZIP13G64D protein is readily degraded by a proteasome‐dependent mechanism Proteasome inhibitor treatments. 293T cells were transfected with WT‐V5 or G64D‐V5 ZIP13 and treated with 10 μM MG132 or 1 μM bafilomycin for 6 h. Cells were lysed in 1% NP‐40 and then separated into soluble and insoluble fractions. Western blotting analysis was performed with an anti‐V5 or anti‐ubiquitin antibody. HeLa cells expressing WT‐V5 or G64D‐V5 (Supplementary Fig S2A) were treated with 10 μM MG132 for the indicated periods. (Upper) Total cell lysates were analyzed by Western blot using an anti‐V5 antibody. (Lower) The hCD8 levels indicate the amount of transfected plasmid DNA (pMX‐WT‐IRES‐hCD8 or pMX‐G64D‐IRES‐hCD8). Cells were analyzed by flow cytometry using APC‐conjugated anti‐hCD8 antibody. Histograms were gated on hCD8‐positive cells. Confocal images of ZIP13. HeLa cells stably expressing the indicated proteins were treated with or without MG132. Nuclei (blue), ZIP13 (green), Golgi (red), and actin (magenta) were stained with DAPI, anti‐V5 antibody, anti‐GM130 antibody, and Phalloidin, respectively. HeLa cells stably expressing the indicated proteins were treated with proteasome inhibitors 10 μM MG132 or 1 μM lactacystin for 6 h, followed by Western blot of whole‐cell lysates using an anti‐V5 antibody. Location of pathogenic mutations in TM1. Amino acid alignment of the TM1 of human ZIP family members. Red: hydrophobic amino acids; blue: acidic amino acids; magenta: basic amino acids; green: hydrophilic amino acids. AE (G340D): amino acid substitution in ZIP4 of AE patients; SCD‐EDS (G64D): amino acid substitution in ZIP13 of SCD‐EDS patients. The 64th amino acid influences ZIP13 protein stability. C‐terminally V5‐tagged ZIP13 expression plasmids with a mutation at position 64 were transfected into 293T cells and analyzed by Western blot using an anti‐V5 antibody. Mutant ZIP13 constructs with an acidic amino acid at position 64. 293T cells were transfected with C‐terminally V5‐tagged ZIP13 expression plasmids, treated with MG132, lysed in NP‐40, separated into soluble and insoluble fractions, and analyzed using an anti‐V5 antibody. Mutant ZIP13 constructs in which glycine 64 was replaced with asparagine (G64N) or glutamine (G64Q). Total cell lysates were analyzed by Western blot using an anti‐V5 antibody. Source data are available online for this figure. Bum‐Ho Bin et al. EMBO Mol Med. 2014;6:1028-1042 © as stated in the article, figure or figure legend