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. 2019 Nov 14;9:16843. doi: 10.1038/s41598-019-53317-9

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© The Author(s) 2019

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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Caskin1 co-localizes with the postsynaptic marker Shank2 protein and regulate the phosphorylation of GluA1 AMPA receptor subunit. (A) Representative images of a Caskin dKO hippocampal neuron overexpressing EGFP and V5-tagged Caskin1 and immunostained for the presence of endogenous Shank2 protein. Arrows indicate mushroom spines. Bars indicate 2 µm. (B,C) Comparison of endogenous PSD-95 and Shank2 levels in C57Bl6/J wild type (WT) and Caskin dKO brain lysates at the age of 3 months. (C) PSD-95 and Shank2 levels were normalized to the corresponding βIII-tubulin intensity values. In case of independent western blots, Caskin dKO ratios were normalised to WT values on the same blot and then normalised values were averaged between the independent Western blots. (D) Brain lysates of wild-type (WT) and Caskin dKO mice were immunoprecipitated with anti-Caskin1 or anti-Shank2 antibodies, and detected by antibodies against Caskin1, Shank2 and PSD-95 (IB). (E–H) Detection and quantification of S845 and S831 phosphorylation and total GluA1 levels in DIV14-16 hippocampal neuronal cultures isolated from wild-type (WT) or Caskin dKO embryos, with our without cLTP treatment. To block NMDA-receptor dependent changes, 50 μM APV was also applied. βIII-tubulin was detected as a loading control. Graphs were calculated from 4–5 independent cultures by normalizing the pS845/total GluA1/βIII-tubulin (F) and pS831/total GluA1/βIII-tubulin (H) ratios to that of the wild-type control samples. All data are displayed as mean ± SEM. Asterisks represent significance compared to wild-type control samples and the $ symbol indicates significant differences between the indicated categories. *p < 0.05, ^$p < 0.05.