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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 disrupts endothelial barrier integrity and differentially affects Cullin-1, -2 and -3. (A) Time course of normalized endothelial resistance of HUVEC monolayers during CSN5i-3 treatment in different concentrations. (B) Normalized endothelial resistance at timepoint 5 hours after CSN5i-3 treatment in different concentrations. *p < 0.05, ***p < 0.001, ****p < 0.0001 compared to control in Dunnet’s post-hoc test of one-way ANOVA (n = 4). (C) Macromolecule passage (Horseradish peroxidase, HRP) across HUVEC monolayers during CSN5i-3 treatment **p < 0.01, ****p < 0.0001 compared to control in Tukey’s post-hoc test of two-way repeated measures ANOVA (n = 3 independent experiments). (D) Western blot analysis of Cullin-1, Cullin-2 and Cullin-3 after 5 hours of CSN5i-3 treatment (1 μM or 4 μM). ERK1/2 was used as loading control. Blot images were cropped for clarity of presentation (full blots are in Supplemental Fig. 3). (E) Quantification of neddylated versus unneddylated Cullin-1, Cullin-2 and Cullin-3 after CSN5i-3 treatment. *p < 0.05, ***p < 0.001 compared to control in Dunnett’s post-hoc test of one-way ANOVA (n = 3 independent experiments). (F) Quantification of total Cullin-1, Cullin-2 and Cullin-3 levels after CSN5i-3 treatment. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control in Dunnett’s post-hoc test of one-way ANOVA (n = 3).