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. 2019 Feb 26;9:2753. doi: 10.1038/s41598-019-39073-w

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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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XPB could anchor SPB2 to the sites of UV damage. (a) Schematic representation of the LacO-tethering assay. (b) Recruitment of SPB2 to the anchored GFP-LacR-NLS or GFP-LacR-XPB or GFP-LacR-XPB∆N is shown in U2OS17 cells. Immunostainings were performed both under normal conditions (NT) and upon UV irradiation (UV 1 h). Arrowheads indicate the cellular position of the LacO arrays. Scale bars represent 5 µm. (c) The values on the graph represent the frequency of the co-localization of SPB2 with GFP obtained from 100 cells in case of each experiment (N = 3). Black columns represent the GFP-LacR-NLS, grey columns represent the GFP-LacR-XPB, while dark grey columns represent the GFP-LacR- XPB∆N data, respectively. (d) Immunoblot detection of SPB2 and ubiquitylated protein interaction in U2OS cells. The success of the immunoprecipitation experiment was controlled with anti-HA antibody and the connection between SPB2 and ubiquitin was detected with anti-GFP antibody (since cells were transfected with plasmid encoding SPB2-EGFP fusion protein) in the precipitated samples both in control and UV treated (2 h and 4 h) samples. Immunoblot detection of GFP and HA signals in the input samples are shown in the lower panel.