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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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NEDD8 must simultaneously substitute the residues 4, 12, 14 and 72 with the Ub equivalents for efficient hydrolysis by USP2. (A) Wild-type NEDD8-His_x6 and indicated mutants (13.8 μM) were incubated with or without USP2 (4.75 μM) at 37 °C for 60 min. All reactions were terminated by adding 4X SDS-PAGE sample buffer and incubating at 100 °C for 10 min. Samples were separated on 16.6% SDS-PAGE and further analyzed using western blotting with the anti-His_x6 tag antibody. Data are representative of three independent experiments. The full-length blots are presented in Supplementary Figure S3. (B) The signal intensity of three independent experiments was measured by a densitometer and processed by ImageJ. The results of the measurement were interpreted as a bar graph. Values are means ± S.D. from three independent experiments. The numbers noted at the bottom represent the lane numbers on the western blot.