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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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Phosphoisotypes of endogenous GSK3β in various cell lines. (a) Dephosphorylation of endogenous GSK3β with alkaline phosphatase (ALP). The CHO-K1 cell extract was incubated with 2.5 U/ml ALP for 3 h and was subjected to Laemmli’s (upper) and Phos-tag (lower) SDS-PAGE, followed by immunoblotting with anti-GSK3β, anti-pS9 and anti-pY216 antibodies. In Phos-tag blot the double-arrowhead indicates Ser9/Tyr216 double phosphorylated GSK3β, the arrowhead indicates Tyr216-phosphorylated GSK3β, and the bar indicates nonphosphorylated GSK3β. (b) Immunoblots showing the endogenous GSK3β in cultured cell lines. The cell extracts of CHO-K1, SH-SY5Y, PC-12, COS-7 and HEK293 cells were subjected to Laemmli’s SDS-PAGE, followed by immunoblotting with anti-GSK3β, anti-phospho-Ser9 (pS9) and anti-phospho-Tyr216 (pY216) antibodies. Actin was used as the loading control. An asterisk in the pY216 blot indicates GSK3α. Phos-tag immunoblot of endogenous GSK3β with anti- GSK3β is shown in the bottom panel. The labeling of three bands in Phos-tag is as in (a). (c) Quantification of the GSK3β phosphoisotypes: 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 = 3). Uncropped immunoblots after Laemmli’s and Phos-tag SDS-PAGE are provided in Supplementary Fig. 6.