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. 2017 Aug 7;7:7405. doi: 10.1038/s41598-017-07565-2

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© The Author(s) 2017

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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TPCA-1 increases GR level and impairs GR down-regulation. (A) TPCA-1 does not change GR phosphorylation level. A549 cells were treated for 30 min with Dex and TPCA-1 as indicated. Immunoblotting analysis was performed to measure GR phosphorylation level. (B) TPCA-1 does not alter Dex induced nuclear translocation of GR. A549 cells were treated as in (A). The cytoplasmic (C) and nuclear fractions (N) were prepared and analyzed using immune- blotting to assay GR level. GAPDH and histone H3 were applied as internal standards for cytoplasmic and nuclear fractions, respectively. (C) TPCA-1 increases GR level. A549 cells were treated with different concentrations of TPCA-1 for 48 h and crude cell extract was immune-blotted to measure GR level. (D) TPCA-1 impairs Dex induced GR down-regulation. A549 cells were treated with Dex and TPCA-1 as indicated, and GR level was analyzed. (E) IKK2 modulates GR level and down-regulation. A549 cells were transfected with Non-targeting (NT) or IKK2 siRNA for 48 h, and then treated with Dex for additional 48 h. Immuno-blotting was used to analyze GR and IKK2 levels. Full-length blots are presented in Supplementary Figure S4.