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. 2017 Jun 12;8:15751. doi: 10.1038/ncomms15751

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Copyright © 2017, The Author(s)

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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(a,b) Mobilization of APC12 (a) and Cdh1 (b) to sites of DNA damage. U2OS cells were transfected with GFP-APC12 or GFP-Cdh1 expression plasmids and the indicated siRNAs, microirradiated with laser and imaged at different time points. (c) Interaction of Cdh1 with Emi1 in S/G2 cells. HeLa cells stably expressing strep-tagged Cdh1 were synchronized with double thymidine, released into S/G2, exposed to 10 Gy IR and collected after 1 h recovery for immunoprecipitation analysis. (d) The accumulation of K11-linked poly-ubiquitin at sites of DNA damage. U2OS cells were transfected with ubiquitin (Ub) constructs, microirradiated and processed 30 min later for immunostaining of His tag and BRCA1. (e) The accumulation of the endogenous K11-linked Ub chains at sites of DNA damage. Immunostaining of K11-linked Ub and γH2AX was performed 30 min after microirradiation. Top: representative images of K11-linked poly-(Ub) and γH2AX immunostaining in U2OS cells transfected with the indicated siRNAs. Bottom: quantification of K11 linkage intensity at microirradiated regions (>110 cells quantificated). Error bars indicate SEM from three independent experiments. ** (in e) indicates P<0.001 (student’s t-test). Scale bars represent 10 μm.