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. 2016 Feb 16;6:21224. doi: 10.1038/srep21224

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Copyright © 2016, Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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Using nuclear-cytosol extraction kits according to the manufacturer’s instructions, cytoplasmic and nuclear fractions were separated by using two of protocols: 1. Adding CEB-B solution to produce the Nucleus1 and Cytoplasm1 fractions containing nuclear membrane proteins; 2. Without adding CEB-B solution to produce the Nucleus2 and Cytoplasm2 fractions without nuclear membrane proteins. Samples (300 μg) of each group were subjected to Co-IP using an anti-Cx43 antibody (3 μg). Immunoprecipitated lysates (100 μg) were then analyzed by Western blotting using anti-AKAP95 and anti-Cx43 antibodies. The experiment was repeated three times.