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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 mouse brains. (a) Expression and Ser9 phosphorylation of GSK3β in mouse brain with aging. Extracts of hippocampus of male and female mouse at ages 3 weeks (3 W), 3 and 6 months (3 M and 6 M), and 1.5 years (1.5Y), were immunoblotted with anti-GSK3β or anti-phospho-Ser9 after Laemmli’s SDS-PAGE. The ratio of phospho-Ser9 to total GSK3β was quantified and expressed to the ratio at 3 weeks. Gray represents male and black represents female (means ± s.e.m. n = 6, *p < 0.05, **p < 0.01, one-way ANOVA). (b and c) Analysis of GSK3β phosphorylation in different regions of brains prepared from 1.5-year-old mice; cerebral cortex (Cx), cerebellum (Cb), olfactory bulb (Ob) and hippocampus (Hc). (b) shows the male mice and (c) shows the female mice. The top four panels show Laemmli’s SDS-PAGE and the lower two panels (upper, shorter exposure; lower, longer exposure) show Phos-tag SDS-PAGE that had been immunoblotted with the indicated antibodies. The percentages of three phosphoisotypes of GSK3β are shown below the blots. Dark gray represents phospho-Ser9 and phospho-Tyr216 GSK3β, light gray represents phospho-Tyr216, and black represents nonphosphorylated GSK3β. The percentages of phospho-Tyr216 are indicated (means ± s.e.m. n = 6, 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. 10.