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. 2023 Jan 10;14:79. doi: 10.1038/s41467-022-35608-4

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© The Author(s) 2023

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. 2 — a Domain structure of each component of the RQT complex. b Schematic of the experiments. c, e, g Purification of the complex containing the indicated RQT factors. Different combinations of the indicated RQT factors were co-expressed in yeast cells. The complex was affinity-purified via Slh1-Flag-TEV-protein A using IgG magnetic beads. Copurified RQT factors were separated by 8% Nu-PAGE and detected by Coomassie brilliant blue (CBB) staining or immunoblotting using an anti-Flag antibody. d, f, h Pull-down assay of the RQT complex with the K63- or K48-linked tetraubiquitin chain. The complex containing the indicated RQT factors was immobilized on IgG magnetic beads and mixed with the K63- or K48-linked tetraubiquitin chain. After binding and washing steps (as indicated in 2b), the proteins in the final elution were separated by 10% Nu-PAGE and detected by CBB staining or immunoblotting using an anti-ubiquitin antibody. All experiments were performed at least twice with highly reproducible results.