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. 2022 Sep 26;13:5644. doi: 10.1038/s41467-022-33285-x

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© The Author(s) 2022

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 The results from the MS analysis were analyzed for enrichment. Among the pathways (P < 10⁶) with the highest degree of enrichment, two were related to the regulation of protein stability (‘Proteasome Degradation’ and ‘Regulation of Protein Stability’). b HEK293FT cells were transfected with Flag-tagged DUBs and HA-IDO1 for 48 h. Lysates were immunoprecipitated with anti-Flag and analyzed. c HEK293FT cells were transfected with HA-USP14 and Flag-tagged IDO1, IDO2, TDO2, or AFMID for 48 h. Lysates were immunoprecipitated with anti-Flag and analyzed. d HCT116 cells were immunoprecipitated with anti-IDO1 or anti-USP14 and analyzed. e Purified USP14 was incubated with GST or GST-IDO1 coupled to GSH-Sepharose, followed by Coomassie blue staining. f, g HCT116 cells were transfected with the indicated constructs (f), and lysates were incubated with GST or GST-IDO1-GSH-Sepharose (g). Proteins retained on Sepharose were blotted with the indicated antibody. h Western blotting analyses and quantification of the indicated cells transfected with USP14 plasmids, or control shRNA and USP14 shRNA. i KYN and TRP levels were measured using ELISA in the supernatants of cells transfected with USP14 plasmids, or control shRNA and USP14 shRNA. j, k HEK293FT cells were transfected with IDO1 and Flag-tagged USP14 or USP14 ΔUBL (j) for 48 h, or HCT116 cells were transfected stably with control shRNA and USP14 shRNA (k) were treated with CHX (0.1 mg mL⁻¹) for the indicated time, and lysates were analyzed. Graph showing the amount of IDO1 protein remaining after CHX treatment as a percentage of the starting IDO1 protein level. l The indicated cells were treated with IU1 (50 µм) or DMSO for the indicated time and lysates were analyzed. m Graph showing the amount of IDO1 protein remaining after IU1 treatment as a percentage of the starting IDO1 protein levels. Error bars represent the mean ± SD of three (h, i) or five (j, k, and m) independent experiments, ns not significant. In h, i, two-sided Student’s t-test. In j, k, and m, One-way repeated-measures ANOVA test. Source data are provided as a Source Data file.