FIGURE 7.

SUMOylation of VCP promotes its hexamer assembly. A and B, increased VCP hexamer assembly in vivo during stress response. The abundance of the VCP hexamers in the liver of mice treated with sodium arsenite (A) or paraquat (B) were analyzed by PAGE under non-denaturing (−SDS) or denaturing (+SDS) conditions (see “Experimental Procedures”). Quantification of the data represents mean ± S.E. n = 3 experiments. C, Ubc9 fusion-directed SUMOylation (UFDS) of VCP promotes the hexamer formation. HEK293 cells were lysed and analyzed as described above. VCP hexamer formation was assessed in a non-denaturing (−SDS) condition, and the total VCP in each sample was determined by SDS-PAGE, and serve as loading control. D, size exclusion chromatography of His-tagged WT VCP and VCP-Ubc9 fused proteins. HEK293 cells were co-transfected with CFP-SUMO1 and the indicated VCP constructs, and the lysates were fractionated using size exclusion chromatography followed by SDS-PAGE and immunoblotting. E, quantification of the data shown in D. Elution peaks of molecular weight markers are shown. Results represent mean ± S.E. n = 3 experiments.