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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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Derivation and characterization of cell lines lacking Atp7a, MtI and MtII genes. (a) Primary fibroblasts were isolated from the lungs of Atp7a^fl/Y;MtI^+/+/MtII^+/+ and Atp7a^fl/Y;MtI^−/−/MtII^−/− mice and then immortalized by transfection with a plasmid expressing the SV40 large T antigen (SV40 Tag) resulting in WT and MT- cells, respectively. An adenoviral vector encoding CRE recombinase was used to delete Atp7a in WT and MT- cells to obtain ATP7A- and ATP7A-/MT- cells, respectively. (b) PCR analysis of genomic DNA was used to confirm deletion of MtI and MtII genes in both the MT- and ATP7A-/MT- cell lines. Expected PCR product sizes: MtI gene (WT = 161 bp; knockout = 176 bp); MtII gene (WT = 282 bp; knockout = 299 bp). (c) Immunoblot analysis was used to confirm the loss of ATP7A protein in both ATP7A- and ATP7A-/MT- cell lines. Tubulin was detected as a loading control. Images of full-length gels and immunoblots are provided in the supplementary data.