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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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Effects of LiCl and SB415286 on the phosphorylation states of GSK3β. (a) Effects of LiCl on the phosphorylation states of GSK3β. CHO-K1 cells were treated with 10 mM LiCl for 5 or 10 h and the resulting cell extracts were subjected to Laemmli’s and Phos-tag SDS-PAGE, followed by immunoblotting with the indicated antibodies. An asterisk in the pY216 blot indicates GSK3α. In Phos-tag (bottom), the double-arrowhead represents Ser9/Tyr216-phosphorylated GSK3β, the arrowhead represents Tyr216-phosphorylated GSK3β, and the bar represents nonphosphorylated GSK3β. Quantification of phospho-Ser9 was shown in the lower graph; gray represents the data from Laemmli and black represents the data from Phos-tag SDS-PAGE (means ± s.e.m. n = 6, **p < 0.01, ***p < 0.001, one-way ANOVA). (b) Phosphorylation of β-Catenin in CHO-K1 cells treated with LiCl as in (a). The CHO-K1 cell extracts were immunoblotted with anti-β-Catenin or anti- phospho-β-Catenin. Actin was the loading control, the same as shown in (a). The ratio of phospho-β-Catenin to total β-Catenin was quantified and expressed against that of the control at 0 h (means ± s.e.m. n = 6, ***p < 0.001, one-way ANOVA). (c) Effects of SB415286 on the phosphorylation states of GSK3β. CHO-K1 cell extracts treated with 20 μM SB415286 for 5 or 10 h were subjected to Laemmli’s (upper four panels) and Phos-tag (bottom) SDS-PAGE, followed by immunoblotting with the indicated antibodies. An asterisk in the pY216 blot indicates GSK3α. In Phos-tag, the phosphoisotypes are indicated as in (a) and are quantified: the upper dark gray section represents phospho-Ser9/phospho-Tyr216, the middle light gray section represents phospho-Tyr216, and the lower black section represents nonphosphorylated GSK3β. The percentages of phospho-Tyr216 are indicated (means ± s.e.m. n = 6, *p < 0.05, **p < 0.01, one-way ANOVA). Uncropped immunoblots after Laemmli’s and Phos-tag SDS-PAGE are provided in Supplementary Fig. 7.