The mutant ZIP13 protein is degraded through a VCP‐dependent mechanism Identification of VCP/Cdc48/p97 as a ZIP13‐associating protein. The mutant ZIP13.

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The mutant ZIP13 protein is degraded through a VCP‐dependent mechanism Identification of VCP/Cdc48/p97 as a ZIP13‐associating protein. The mutant ZIP13 protein is degraded through a VCP‐dependent mechanism Identification of VCP/Cdc48/p97 as a ZIP13‐associating protein. Whole‐cell lysates from 293T cells transfected with FLAG‐tagged ZIP13 were immunoprecipitated with an anti‐FLAG antibody, followed by SDS–PAGE and silver staining. Unique bands were cut out and analyzed by TOF/MASS to identify the proteins. A protein band near 88 kDa was determined to be VCP/Cdc48/p97. VCP was also detected by Western blot using an anti‐VCP antibody (lower). IgH: heavy chain of IgG; IgL: light chain of IgG; A: SP‐uncleaved immature ZIP13 protein; B: SP‐cleaved mature ZIP13 protein. VCP binds to ZIP13. Whole‐cell lysates from 293T cells transfected with expression plasmids for V5‐tagged ZIP13 proteins were immunoprecipitated with an anti‐V5 antibody, followed by SDS–PAGE. VCP and ZIP13 proteins were detected by Western blot using anti‐VCP and anti‐V5 antibodies, respectively. The VCP/ZIP13 ratio was analyzed using ImageJ software (http://rsbweb.nih.gov/ij/download.html) (bottom). Confocal images of VCP in HeLa cells stably expressing G64D‐V5. VCP (green) and G64D‐V5 (red) were stained with anti‐V5 and anti‐VCP antibodies, respectively. Effect of VCP siRNA on the protein expression of G64D‐V5 in HeLa cells. VCP siRNA was transfected into HeLa cells stably expressing G64D‐V5. Seventy‐two hours posttransfection, the cells were harvested and subjected to Western blotting analysis using anti‐V5 or anti‐VCP antibodies. Effect of a dominant‐negative form of VCP on the protein expression of G64D‐V5 in HeLa cells. 3xFLAG‐tagged wild‐type VCPWT and dominant‐negative VCPE305Q/E578Q were transfected into HeLa cells stably expressing G64D‐V5. Twenty‐four hours later, the cells were lysed and then subjected to Western blotting analysis with anti‐V5 or anti‐FLAG antibodies. Effect of a VCP inhibitor, DBeQ on the protein expression of G64D‐V5 in HeLa cells. HeLa cells stably expressing WT‐V5 or G64D‐V5 were treated with 10 μM MG132 or 10 μM DBeQ together with CHX for the indicated times. The cell lysates were subjected to Western blotting analysis with an anti‐V5 antibody. Right graph shows the relative expression level of ZIP13 proteins. Data are representative of two independent experiments. 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