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. 2019 Jul 4;9:9647. doi: 10.1038/s41598-019-46250-4

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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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Increase in plasma membrane surface localization of the D97S mutant by endocytosis inhibitor or primaquine treatment. (A,B) MDCK cells stably expressing the FLAG-tagged D97S mutant were treated with MDC and MβCD for 3 h. After collecting cell lysates, the aliquots were blotted with anti-FLAG and anti-β-actin antibodies. The band densities of proteins are represented relative to the values with 0 μM. (B) The cells were stained with anti-FLAG (CLDN16) and anti-ZO-1 antibodies. (C,D) MDCK cells stably expressing the FLAG-tagged D97S mutant were incubated with primaquine (PQ, 100 μM). The biotinylated proteins were blotted with anti-FLAG and anti-CLDN1 antibodies. (E) The cell lysates of MDCK cells expressing the WT or D97S mutant were blotted with anti-FLAG and anti-β-actin antibodies. The band densities of proteins are represented relative to the values at 0 h. The full-length blot images are shown in Supplementary Figs S6–S8. (F) The cells were incubated with anti-FLAG (CLDN16) and anti-ZO-1 antibodies. The scale bar indicates 10 μm. n = 4 in four independent experiments. **P < 0.01 and *P < 0.05 compared with 0 μM.