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. 2018 Mar 29;9:1285. doi: 10.1038/s41467-018-03588-z

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — USP52 is a deubiquitinase. a In vitro deubiquitination assays with E. coli cells-purified UCH domain of USP52 (USP52/UCH, 1 μg) and K48-linked tetra-ubiquitin linkages (1 μg) under different time points as indicated. The cleavage effect was examined by western blotting with antibody against ubiquitin. The asterisk indicates the recombinant protein stained by Commassie Blue. b In vitro deubiquitination assays with Sf9 cells-purified USP52/UCH (1 μg) and K48-linked tetra-ubiquitin linkages (1 μg) under different time points as indicated. The cleavage effect was examined by western blotting with antibody against ubiquitin. The asterisk indicates the recombinant protein stained by Commassie Blue. c In vitro deubiquitination assays with different types of ubiquitin linkages and increasing amounts of Sf9 cells-purified USP52/UCH (1 and 3 μg) or control eluents. After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. d In vitro deubiquitination assays with different types of ubiquitin linkages (1 μg) and HeLa cells-purified full-length USP52 (1 μg) or UCH domain deficient USP52 (USP52/∆UCH, 1 μg) with high salt and detergent buffer. After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. The asterisk indicates the recombinant protein stained by silver staining. e In vitro deubiquitination assays with K48- or K63-linked tetra-ubiquitins (0.5 μg) and Sf9 cells-purified USP52/UCH (3 μg) in the presence or absence of 2 mM alkylating reagent N-ethylmaleimide (NEM). Sf9 cells-purified recombinant C-terminal USP7 (UCH domain and C-terminal ubiquitin-like domain, 1.5 μg) was taken as a positive control. After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. f In vitro deubiquitination assays with K48- or K63-linked tetra-ubiquitins (0.5 μg) and Sf9 cells-purified USP52/UCH (3 μg) or cysteine mutant of UCH domain (C528A/C530A, 3 μg). After 4 h of incubation, the cleavage effect was examined by western blotting with antibody against ubiquitin. CA represents mutant carrying both C528A and C530A (C528A/C530A)