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. 2021 Apr 6;12:2043. doi: 10.1038/s41467-021-22319-5

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© The Author(s) 2021

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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Fig. 6 — a Schematic of in vitro degradation assay. b In vitro 35S-FBW7α degradation assay performed in cell-free extracts from cells of the indicated genotypes and visualised by autoradiography. c In vitro FBW7 degradation assay performed in cell-free extracts in the presence of the indicated ubiquitin mutants and visualised by autoradiography. d Schematic for in vitro ubiquitylation and ubiquitin restriction enzyme digest experiment for 35S-FBW7. e Autoradiographs showing cleavage of beads-bound polyubiquitylated 35S-FBW7 by the indicated DUBs. Linkage specificity is given in parentheses. Black stars (*) in b, c, and e represent the nonspecific band of unknown identity. f Silver stain on cleaved ubiquitins in the supernatant from the experiment in e, black stars represent respective DUBs and red stars label uncleaved ubiquitin oligomers. All blots are representative of at least three independent experiments with the exception of e, which was done twice with similar results. Source data are provided as a Source Data file.