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. 2023 Dec 15;16(1):10–39. doi: 10.1038/s44321-023-00003-z

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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 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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(A, B) Western blot (A) and qPCR (B) analysis in T47D/MCF-7 cells infected with shCtrl or shMYSM1 lentivirus to evaluate the impact of MYSM1 depletion on ERα protein and mRNA levels. (C) MCF-7 cells carrying control shRNA or shMYSM1 were complemented with CHX (20 μM) for particular time periods, and cell lysates were assessed by western blot. Relative ERα level was quantified by densitometry and presented in the right plot. (D) Western blot analysis of ERα and exogenous MYSM1 expression in MCF-7/HEK293 cells transfected with wild-type MYSM1 plasmid or MPN-deletion mutant. (E) Cell lysates from MYSM1-FL/MYSM1-ΔMPN overexpressing MCF-7 cells stimulated with CHX (20 μM) at specified time points were denatured and quantitated by western blot. Relative ERα level was quantified by densitometry and presented in the right plot. (F, G) Extracts obtained from MCF-7 cells harboring shMYSM1 or FLAG-MYSM1 upon MG132 (10 μM) treatment for 4 h were analyzed by western blot. (H) MCF-7 cells from the control or the MYSM1-knockdown group were harvested after MG132 (10 μM) addition for 6 h, followed by immunoprecipitation with anti-ERα and subsequently probed with anti-His. (I) Immunoblot analysis of the polyubiquitination of ERα proteins in MCF-7 cells transiently co-transfected with plasmids encoding ERα and FLAG-MYSM1 or MYSM1-ΔMPN. MG132 (10 μM) was added to cell culture 6 h before cell collection. Cell lysates were subjected to immunoprecipitation with anti-ERα and immunoblot with anti-His. (J, K) HEK293 cells transfected with the indicated plasmids were extracted and immunoprecipitated with anti-ERα, followed by immunoblot with anti-HA. Data information: (A–K): n = 3 independent experiments performed in duplicate. (A, B, D): mean ± SD; Student’s t test; n = 3, NS: not significant. Source data are available online for this figure.