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. 2024 Jan 2;43(2):250–276. doi: 10.1038/s44318-023-00018-9

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© The Author(s) 2024

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 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/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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Figure EV1 — (A) FLAG-WT USE1, FLAG-USE1 ΔPolyAla, FLAG-USE1 C188A, and FLAG-USE1 ΔLB were co-expressed with HA-Ub in HEK293T cells. Cell lysates were incubated with or without β mercaptoethanol (βME) and analyzed for ubiquitin loading. Results are normalized to control WT USE1. n = 4 biological replicates. (B) A representative blot for time-dependent in vitro ubiquitin loading of WT and ΔPolyAla USE1 by UBA6 (quantification is presented in Fig. 1C). (C) WT and ΔPolyAla KO cells were treated with the cross-linker formaldehyde and cell lysates were immunoprecipitated with anti-USE1 or control IgG antibodies. Immunocomplexes were analyzed with anti-USE1 and anti-UBA6 antibodies. (D) FLAG-WT USE1, empty GFP and GFP-polyAla (19Ala) constructs were transfected into HEK293T cells. Cell lysates were immunoprecipitated with anti-UBA6 antibodies and the immunocomplexes were analyzed with anti-FLAG, anti-UBA6 and anti-GFP antibodies. The bound USE1/UBA6 ratio is shown. n = 3 biological replicates. Data information: Data points in (A, D) represent mean ± s.e.m. P values were calculated by one-way ANOVA Tukey’s test (A) or paired 2-tailed t test (D). **P< 0.01, ****P < 0.0001. Source data are available online for this figure.