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. 2017 May 23;7:2304. doi: 10.1038/s41598-017-02322-x

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© The Author(s) 2017

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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USP21 displayed the same substrate recognition mechanism as USP2. Ub, Ub^{4K/12E/14E/72A}, NEDD8, and NEDD8^{4F/12T/14T/72R} (13.8 μM) were incubated with or without USP21 (4.425 μM) at 37 °C for 60 min. Samples were separated on 16.6% SDS-PAGE and further analyzed using western blotting with the anti-His_x6 tag antibody.