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. 2022 Jan 13;18(4):422–431. doi: 10.1038/s41589-021-00952-x

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© The Author(s) 2022

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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Extended Data Fig. 8 — (a) Synthesis and purification scheme to generate SMAC-K48Ub_n from multiple-mono Ub primed SMAC-Ub_1-3. (b) His-TEV-Ub, UBE2R1, and BIRC2 were reacted with SMAC-Ub_1-3 and varying lengths of SMAC-K48Ub_n are isolated using (c) final Superdex 75 gel filtration step. (d) Non-reduced SDS-PAGE showing ubiquitination of SMAC-K48Ub_3-6 (Fraction 3 from b,c) catalyzed by indicated UBE2K variants. Asterisks (*) indicate fluorescent-labeled Ub. (e) Plots of Ub^* transferred to SMAC-K48Ub_3-6 from d, data normalized to UBE2K^WT and presented as mean value ± SD from three independent experiments (n = 3).

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