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. 2017 Aug 17;7:8602. doi: 10.1038/s41598-017-09239-5

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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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GSK3β phosphoisotypes in the brains of db/db mice. (a) GSK3β, phospho-Ser9 (pS9), phospho-Tyr216 (pY216) and actin in the cerebral cortex of wild type mice (+/+, left two lanes) or db/db (right two lanes) were examined using the Laemmli’s SDS-PAGE and phosphorylation of GSK3β by Phos-tag method. (b) The ratio of phospho-Ser9 to total GSK3β in wild type (+/+) and db/db mice. The left two bars show the data obtained from the anti-phospho-Ser9 immunoblot after Laemmli’s SDS-PAGE and the right two bars show the data obtained with Phos-tag SDS-PAGE. The ratio is expressed relative to that in the wild type (+/+) mouse brain (means ± s.e.m. n = 4, *p < 0.05, **p < 0.01, Student’s t test). (c) The percentages of the GSK3β phosphoisotypes on Phos-tag SDS PAGE were quantified: dark gray, phospho-Ser9/Tyr216; light gray, phospho-Tyr216; and black, nonphosphorylated GSK3β. The percentages of phospho-Tyr216 are indicated (means ± s.e.m. n = 4, ns, not significant). Uncropped immunoblots of GSK3β, pS9, pY216 and actin after Laemmli’s SDS-PAGE and GSK3β after Phos-tag SDS-PAGE are provided in Supplementary Fig. 11.