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. 2024 Jan 17;24:33. doi: 10.1186/s12935-024-03224-5

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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/. 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 — USP29 interacts with AURKB and removes its K48-linked polyubiquitination. (A) Immunoprecipitation of Flag-USP29 using anti-Flag antibody. Total cell proteins extracted from MGC-803 cells expressing Flag-tagged USP29 or vector alone were subjected to immunoprecipitation using anti-Flag beads, which were resolved by SDS-PAGE and visualized by silver staining. (B) Liquid chromatography-tandem mass spectrometry analysis of the Flag-USP29-associated peptides corresponding to AURKB. (C) 293T cells overexpressing Myc-USP29 and/ or Flag-AURKB were subjected to reciprocal Co-IP to detect protein interaction. (D) Lysates from MGC-803 and HGC-27 cells were subjected to immunoprecipitation using AURKB antibodies, and USP29 was detected by immunoblot. (E) Left: A schematic representation of various Flag-USP29 truncations; right: lysates from 293T cells overexpressing HA-AURKB and respective Flag-USP29 truncation were subjected to Co-IP and immunoblot. (F) 293T cells were co-transfected with HA-AURKB, Myc-Ub and Flag-USP29/USP29 C294S mutant (CS), and polyubiquitination analysis of AURKB was shown. G and H. 293T cells were co-transfected with indicated Flag-AURKB, Myc-USP29, HA-Ub (WT) and various HA-Ub mutant plasmids, polyubiquitination analysis of AURKB was analyzed. The experiments were independently repeated three times with similar results (A, C-D, right of E, F–H)