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. 2019 May 31;9:8131. doi: 10.1038/s41598-019-44595-4

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© The Author(s) 2019

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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CSN5i-3- induces transcription of RhoB mRNA. (A) HEK293T cells were cotransfected with mCherry-RhoB and HA-ubiquitin and treated with CSN5i-3 (1 and 4 μM) or MLN4924 for five hours, with addition of MG132 for the last four hours. Next, HA-ubiquitin was immunoprecipitated under denaturing conditions. Samples were analyzed by western blot for presence of RhoB and ubiquitin using RhoB and HA antibodies, respectively. GAPDH was used as loading control. Blot images were cropped for clarity of presentation (full blots are in Supplemental Fig. 8). (B,C) RNA expression, determined by qPCR, of RhoA (B) and RhoB (C) from HUVECs lysates after 5 hours of treatment with CSN5i-3 (1 and 4 μM) or 10 ng/ml TNF-α *p < 0.05, ***p < 0.001, ****p < 0.0001 after Dunnet’s post hoc analysis of one-way ANOVA (n = 3). (D–E) Western blot analysis and quantification of RhoB expression after CSN5i-3 treatment (1 and 4 μM) for 0, 1, 5, 8 and 16 hours, with or without pre-treatment of 0.5 μg/ml cycloheximide. Blot images were cropped for clarity of presentation (full blots are in Supplemental Fig. 9). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 after Dunnet’s post hoc analysis of two-way ANOVA (n = 3).