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. 2014 Aug 12;289(39):27278–27289. doi: 10.1074/jbc.M114.589812

FIGURE 6.

FIGURE 6.

USP7 deubiquitinates XPC-ubiquitin conjugates in vitro. Deubiquitination assays were performed using soluble chromatin (Sol.Chr.) factions isolated from the UV-irradiated HCT116-USP7−/− cells and recombinant His-tagged USP7 (His-USP7) or the USP7 catalytic domain (USP7-CD) in DUB buffer. The reaction mixtures, containing ∼6 μg of soluble chromatin and the indicated amount of His-USP7 or USP7-CD, were incubated at 37 °C and stopped at 120 min. XPC and its ubiquitin conjugates were detected by Western blotting for XPC. The asterisk indicates a nonspecific protein band as in Fig. 1D. A, His-tagged USP7. B, the USP7 catalytic domain. C, time course of the deubiquitination reaction with 0.2 μm His-tagged USP7. D, inhibition of USP7-mediated XPC deubiquitination by HBX 41108. Lane 1L shows lane 1 at a lower exposure. E, XPC deubiquitination assays were performed using affinity-purified WT-USP7 or CS-, M1-, and M2-USP7 mutant proteins. (UB)n, multiple (n) Ub moieties.