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. 2014 Jul 21;206(2):183–197. doi: 10.1083/jcb.201311063

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© 2014 Zeman et al.

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Figure 3. — Rad18 is deubiquitinated after MMS treatment. (A) Endogenous Rad18 is monoubiquitinated. Rad18 was immunoprecipitated (RF antibody) from untransfected cells, and purified protein on beads was treated with or without the Usp2 catalytic core (USP2cc) before being analyzed by Western blotting for the presence of ubiquitin. (B) Rad18 is deubiquitinated after MMS treatment. Cells mock transfected or transfected with His-tagged ubiquitin were treated with MMS (0, 25, and 50 ppm) for 4 h before being lysed under denaturing conditions. His-ubiquitin (Ub) and covalently bound proteins were purified on nickel agarose beads (NTA, nitrilotriacetic acid) and analyzed by Western blotting. (C) Proteasome inhibition does not prevent Rad18 deubiquitination after MMS. Untransfected cells were mock or MMS (0.005%) treated in conjunction with 50 µM MG132 or 25 µM LLnL for 4 h and processed as in Fig. 2 C.