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. 2020 May 12;10:7856. doi: 10.1038/s41598-020-64521-3

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© The Author(s) 2020

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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Selection for Cu tolerance in the absence of ATP7A increases ATP7B expression. (a) Immunoblot detection of ATP7B in ATP7A-/MT- cells that were selected for survival in either basal media (Basal) or media containing 20 µM Cu (CuR20). The control sample is ATP7A-/MT- cells propagated in medium containing 50 µM BCS. Each cell line was grown for 16 h in basal media prior to immunoblot analysis. Tubulin was detected as a loading control. (b) Densitometry analysis of ATP7B protein levels normalized against control samples (mean ± SEM; *p < 0.05; ****p < 0.0001). Data were calculated from at least 3 independent experiments. (c) Immunoblot detection of ATP7A and ATP7B proteins in parental ATP7A + /MT- cells (Basal) or ATP7A + /MT- cells that were selected in media containing 900 µM Cu (CuR900). Both cell lines were grown for 16 h in basal media prior to immunoblot analysis. Tubulin was detected as a loading control. (d,e) Densitometry analysis of ATP7A and ATP7B protein levels normalized against Basal samples (mean ± SEM; *p < 0.05; ***p < 0.001). Data were calculated from at least 3 independent experiments. Images of full-length immunoblots are provided in the supplementary data.