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. 2019 Aug 13;9:11783. doi: 10.1038/s41598-019-48286-y

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© The Author(s) 2019

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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(WDR23) isoforms differentially ubiquitinate (GEN1). (A) In the presence of the necessary components for a successful ubiquitination reaction (CUL4A-DDB1-WDR23 complex, UBE1, UBE2D1, ubiquitin, and ATP) GEN1 receives the addition of ubiquitin in a time dependent manner. In the absence of GEN1, less ubiquitination is observed, suggesting minor auto-ubiquitination of the CUL4A-DDB1-WDR23 complex or another unidentified substrate being modified. With the addition of FLAG-GEN1, an increase in the “smear” of ubiquitin is observed over time. Immunoprecipitated samples of GFP-WDR23 isoforms and FLAG-GEN1 were incubated in ubiquitin reaction mixtures and samples were collected at the given time points (0-, 5-, 10-minutes). (B) Modified ubiquitin mutant with all lysine mutated to arginine to prevent the formation of chains. (C) WDR-23A and WDR-23B differentially modify GEN-1 in vitro. Immunoprecipitated samples of GFP-WDR-23 isoforms and GEN-1-FLAG were incubated in ubiquitin reaction mixtures and samples were collected at the given time points (0-, 5-, 10-minutes). αGFP probed for GFP-WDR-23/GFP-WDR23 isoforms. αFLAG for GEN-1-FLAG/FLAG-GEN1. αUbiquitin for ubiquitinated-GEN1/GEN-1.