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. 2019 Jul 17;9:10329. doi: 10.1038/s41598-019-46949-4

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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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Inhibition of p97 leads to substantial accumulation on chromatin-bound substrates. (A) pDNA was incubated in extract supplemented with buffer, the p97 inhibitor CB-5083 (p97i), or p97i and the de-ubiquitinase inhibitor UbVS. DNA-bound proteins were isolated by plasmid pull-down at various times and visualized by Western blot with the indicated antibodies. (B,C) Relative quantitation of DNA-bound p97 (B) and DNA-bound ubiquitin (C) from three biological replicates. Values are normalized to peak accumulation in the +Buffer reaction and error bars represent +/− one standard deviation. (D) pDNA was incubated in extract supplemented with p97i and either wild-type ubiquitin (Ub) or K48R mutant ubiquitin (Ub-K48R). DNA-bound proteins were isolated by plasmid pull-down at various times and visualized by Western blot with the indicated antibodies. Full Western blot images are available in Fig. S1.