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. 2018 Jan 23;8:1405. doi: 10.1038/s41598-018-19538-0

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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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The action of overexpressed GFP-Ub on mHtt IBs is not equivalent to TAMRA-Ub. (a) Scheme of GFP-Ub versus TAMRA-Ub. (b) Confocal images of transient transfected Neuro-2A cells co-expressing Htt-exon1-97Q-C4 and GFP-Ub. 48 hours after transfection cells were ReAsH-stained and fixed for imaging. Scale bar: 5 µm. (c) Insoluble fraction of Neuro-2A cell lysate 48 hours after transient co-transfection of cells with Htt-exon1-97Q and either GFP or GFP-Ub. Formic acid-dissolved aggregates were loaded on a SDS-PAGE and immunoblotted with the antibodies anti-polyQ and anti-GFP. A band of the size of mono-ubiquitinated GFP-Ub-mHtt was detected (arrow) next to GFP-Ub in a high molecular range (asterisk). (d) FRAP analysis of GFP-Ub in cytoplasmic IBs of Neuro-2A cells 48 hours after transfection. Fluorescence recovery of GFP-Ub on cytoplasmic Htt IBs was measured over time. Red semi-circle indicates where bleaching was directed. White rectangle indicates non-bleached area used as a control for photobleaching and normalization. Scale bar: 5 µm. (e) Analysis of fluorescent recovery in bleached areas of Htt IBs indicates a higher mobility of TAMRA-Ub versus GFP-Ub over time. (f) Quantitative analysis reveals a significantly higher fluorescent recovery of TAMRA-Ub compared to GFP-Ub 20 min after photobleaching. ***p < 0.001. Means and SD are shown.