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. 2018 Jan 8;8:68. doi: 10.1038/s41598-017-18513-5

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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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Auto-ubiquitination assays of RBRs with UbcH7 and UbcH5A. Coomassie-stained reducing SDS-gels of auto-ubiquitination assays for HHARI-RBR (A), HOIP-RBR (B), TRIAD3A-RBR (C) and RNF144A-RBR (D). The concentration of RBR used was 5 μM, whilst the concentrations of E1, E2 and Ub were 0.1, 2 and 50 μM, respectively. 10 mM ATP was added to start the reaction and samples were taken after 10, 30 and 60 minutes incubation at room temperature. Red and blue arrowheads indicate UbcH7 and UbcH5A, respectively; black arrowheads indicate E1 (Uba1), E3 and ubiquitin (Ub). In the case of TRIAD3A accumulation of di-ubiquitin (di-Ub) is observed, indicated by a black arrowhead.