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. 2014 Oct 29;42(21):13110–13121. doi: 10.1093/nar/gku1034

Figure 2.

Figure 2.

USP20 deubiquitinates and stabilizes Claspin. (A and B) HEK293T cell lysates were subjected to immunoprecipitation with control IgG or anti-USP20 (A) or anti-Claspin (B) antibodies. The immunoprecipitates were then blotted with the indicated antibodies. (C) Purified His-Claspin was incubated with GST or GST-USP20 coupled to GSH-Sepharose. Proteins retained on Sepharose were then blotted with the indicated antibodies. (D) A549 cells infected with lentivirus encoding the indicated shRNAs were left untreated or treated with MG132 for 4 h. Cells were lysed and the cell lysates were then blotted with the indicated antibodies. (E) A549 cells stably expressing control or USP20 shRNA were transfected with empty vector, shRNA resistant HA-USP20WT or its CA mutant. After 48 h, cells were lysed and cell lysates were then blotted with the indicated antibodies. (F) A549 cells stably expressing control shRNA or USP20 shRNAs were treated with CHX (0.1 mg/ml), and harvested at the indicated times. Cells were lysed and cell lysates were then blotted with the indicated antibodies. Lower panel: quantification of the Claspin protein levels relative to β-actin. (G) Cells transfected with the indicated constructs were treated with MG132 for 4 h before harvest. Claspin was immunoprecipitated and immunoblotted with the indicated antibodies. (H) Cells stably expressing Ctrl or USP20 shRNAs were treated with MG132 for 4 h before harvest. Claspin was immunoprecipitated and immunoblotted with the indicated antibodies. (I) Deubiquitination of Claspin in vitro by USP20. Ubiquitinated Claspin was incubated with purified USP20 or USP20CA in vitro, and then blotted with the indicated antibodies.