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. 2021 Oct 8;40:315. doi: 10.1186/s13046-021-02120-4

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© The Author(s) 2021

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — MUS81 regulates β-TRCP-mediated WEE1 ubiquitination. a and b PCR analysis of MUS81 and WEE1 after MUS81 knockdown in gastric cancer cell lines SGC7901 and BGC823. Data are presented as the mean ± SD (n = 3). c and d Western blot analysis of the impact of MUS81 knockdown on the expression of WEE1 at the protein level in gastric cancer cell lines SGC7901 and BGC823. e Representative images of WEE1 protein degradation after exposure of wild-type MUS81 (MUS81-WT) overexpression and parental AGS cells to cycloheximide (CHX; 50 μg/mL) for different time periods (top) and statistical line graph (bottom). Data are presented as the mean ± SD (n = 3). f Western blot analysis of the effect of chloroquine (10 μM) or MG132 (10 μM) on the degradation of WEE1 protein in MUS81-WT overexpression and parental AGS cells. G Ni-NTA pulldown analysis of 293 T cells transfected with the indicated plasmid to evaluate the effect of MUS81 on the ubiquitination of WEE1. His-Ub, His-tagged ubiquitin; oeMUS81-WT, FLAG-tagged wild-type MUS81; IB WEE1, immunoblotting with anti-WEE1 antibody. h Sketch map of MUS81 enzymatic mutant plasmid construction. Site-directed mutagenesis was used to replace two aspartic-acid residues within the MUS81 nuclease domain (338 and 339) with alanine residues. i Immunoblot of AGS cells transfected with the indicated plasmid, oeVector, FLAG-tagged wild-type MUS81 (oeMUS81-WT), and FLAG-tagged enzymatically inactivated MUS81 (oeMUS81-mut). j Co-immunoprecipitation was performed to detect the effect of MUS81 on the binding of E-3 ligase β-TRCP and WEE1