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. 2023 May 18;30(7):1710–1725. doi: 10.1038/s41418-023-01173-6

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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. 3 — a U2OS cells were transfected with gRNAs targeting USP28 (KO USP28) and parental controls were treated with 50 µg/ml cycloheximide for the indicated times. Levels of USP28 as well as full length (flSREBP2) and mature SREBP2 (mSREBP2) were determined by immunoblotting. Actin is shown as loading control. b Quantification of full-length and mature SREBP2 signals relative to actin from (a). c A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated with 0.5 µg/ml of doxycycline (DOX) or solvent (ethanol, EtOH) for 72 h. During the last 6 h, 20 µM MG-132 was added. Cells were lysed and analysed for expression of full-length (flSREBP2) and mature SREBP2 (mSREBP2). Actin is shown as loading control. d U2OS cells were transfected with expression vectors coding for HA-tagged mature SREBP2 (aa 1-484, HA-mSREBP2) together with HA-tagged wild type (WT) or catalytically inactive (CA) USP28. Levels of USP28 and mature SREBP2 were determined by immunoblotting. Actin is shown as loading control. e HEK293 cells were transfected with expression vectors coding for HA-tagged USP28 or HA-tagged mSREBP2 together with His6-Ubiquitin (K48-only). Ubiquitinated proteins were purified using Ni-NTA and analysed by immunoblotting (Ub). Presence of USP28 and SREBP2 was confirmed in the input. f A431 cells expressing inducible shRNA sequences targeting USP28 (shUSP28#2) were treated with 1 µg/mL doxycycline (DOX) or solvent (ethanol) for 96 h with 20 µM MG132 being added during the last 6 h. Cells were lysed and subjected to immunoprecipitation using antibodies detecting SREBP2. Precipitates were analysed for the presence of ubiquitinated proteins (Ub). Efficient silencing of USP28 and presence of full length and mature SREBP2 was confirmed in the input. Actin is shown as a loading control. IgG bands are marked by asterisks. g U2OS cells were transfected with either wild type or CPD mutant (Ser 432 and 436 to Ala) HA-tagged mature SREBP2 together with HA-tagged USP28 or Flag-FBXW7. Levels of USP28, FBXW7 and mature SREBP2 were determined by immunoblotting. Actin is shown as loading control.