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. 2023 May 12;42:121. doi: 10.1186/s13046-023-02694-1

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© The Author(s) 2023

Open AccessThis 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. 1 — USP3 deubiquitinates REST and extends its half-life. (A) SH-SY5Y cells were treated with the indicated concentrations of proteasomal inhibitor (MG132), lysosomal inhibitor (CQ), or inhibitor of ubiquitin activating enzyme (TAK-243) for 6 h prior to harvest. Immunoblotting was performed with the indicated antibodies. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band (REST/GAPDH) and presented below the blots. (B) SH-SY5Y cells were transfected with increasing concentrations of Flag-USP3 and treated with MG132 for 6 h prior to harvest. A TUBEs assay was performed to assess the ubiquitination status of the REST protein in mock control and Flag-USP3 transfected cells. Cell lysates were immunoprecipitated with TUBEs antibodies, followed by immunoblotting with the indicated antibodies. (C) The ubiquitination and deubiquitination of endogenous REST were analyzed by transfecting SH-SY5Y cells with Flag-USP3, Flag-USP3CS, or sgRNA targeting USP3 followed by immunoprecipitation (IP) with an anti-REST antibody and immunoblotting with an anti-ubiquitin antibody. The cells were treated with MG132 for 6 h prior to harvest. (D) The ubiquitination and deubiquitination of ectopically expressed Myc-REST were analyzed by transfecting HEK293 cells with Flag-USP3 and Flag-USP3CS (E) Flag-USP3 and sgRNA targeting USP3(F) treatment of DUB-inhibitor PR-619 for 48 h in the HEK293 cell line prior to harvest, followed by IP with a Myc antibody and immunoblotting with an anti-ubiquitin antibody. The relative protein expression of REST-(Ub)n with respect to input REST for (C-F) was quantified using ImageJ software and represented as (REST-(Ub)n/REST) below the blot. (G-H) The knockdown effect of USP3 and reconstitution of either (G) Flag-USP3 or (H) Flag-USP3CS in USP3 depleted neuroblastoma on the half-life of endogenous REST in SH-SY5Y cells. CHX (150 µg/mL) was administered for the indicated time, and the cells were then harvested for western blotting with the indicated antibodies