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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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Effect of IGF-1 on GSK3β activity in cultured primary neurons. (a) Effect of IGF-1 on Ser9 phosphorylation of GSK3β in mouse cortical neurons. Neurons in culture at 5 DIV were treated with 100 ng/ml IGF-1 for the indicated times, and the extracts were subjected to Laemmli’s SDS-PAGE, followed by immunoblotting with an anti-GSK3β, anti-phospho-Ser9 and anti-phospho-Tyr216 or Phos-tag SDS-PAGE, followed by immunoblotting with the anti-GSK3β. Actin is the loading control. An asterisk in the pY216 indicates GSK3α. Double arrowhead, Ser9/Tyr216-phosphorylated GSK3β; arrowhead, Tyr216-phosphorylated GSK3β; bar, nonphosphorylated GSK3β. (b) Levels of pSer9 in neurons that were treated with IGF-1 for 30 min. The ratio of pS9/GSK3β was measured and expressed to that of the control at 0 min. The measurements by Laemmli’s and Phos-tag SDS-PAGE are shown in left and right, respectively. (c) The quantification of three phosphoisotypes of GSK3β in a Phos-tag blot: dark gray, phospho-Ser9/phospho-Tyr216; light gray, phospho-Tyr216; black, 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. 9b.