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. 2018 Mar 15;8:4625. doi: 10.1038/s41598-018-23056-4

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© The Author(s) 2018

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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Ascl1 is ubiquitylated by Huwe1 in the cytoplasm. (A) Schematic representation of experimental design. Adenovirally-mediated Cre expression was used to delete Huwe1 from NSCs harbouring the Flox-Huwe1 allele and cells were harvested after 1 day in differentiation media. (B) Flox-Huwe1 NSCs at day 5 were incubated +/− MG132 for 2 hours, and cytoplasmic and chromatin fractions were processed for western blot analysis to detect Huwe1 as well as α-Tubulin (loading control for cytoplasmic fraction) and histone H3 (loading control for chromatin fraction). (C) Cytoplasmic fractions with and without cre-mediated knockdown of Huwe1 (see panel B), were incubated with and without MG132 for 2 hours and western blotted to detect free and ubiquitin-conjugated (blue bracket) forms of Ascl1. Top panel is long exposure to reveal high molecular weight ubiquitylated forms of Ascl1 (blue bracket), bottom Ascl1 panel is lower exposure (cropped from the same blot) to show stabilisation of Ascl1 after Huwe1 knockdown, α-Tubulin (loading control for cytoplasmic fraction, cropped to show specific band of correct molecular weight), n = 3.