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. 2018 Sep 10;8:13489. doi: 10.1038/s41598-018-31657-2

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© The Author(s) 2018

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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p300G422S-KI shows the impaired CREB binding to p300 at the age of 6 but not 3 months. (a) Co-immunoprecipitation analysis of the interaction between p300 and CREB in the hippocampi of WT and p300G422S-KI mice at 3 and 6 months. Hippocampal lysates were immunoprecipitated with a p300 antibody, washed and blotted with the indicated antibody. Arrow indicates CREB expression band. (b) The graph indicates the fold changes of the relative pulled-down CREB protein over the total p300 amounts, as determined by densitometry and normalized to samples from 3 months WT, (two-way ANOVA: F (1,16) = 4.86, p = 0.04; Tukey’s multiple comparisons, *p < 0.05, n = 5). (c) Co-immunoprecipitation analysis of the interaction between p300 and CREB in C57B6 strain hippocampi at the age of 3 and 6 months as described in (a). The graph data was normalized to one of 3 months old C57B6 samples. (d) Western blot analysis for pS133-CREB and pT410/403-aPKC zeta/iota in hippocampal extracts from 3 and 6 months WT and p300G422S-KI mice. Blots were reprobed for total CREB or aPKC as loading controls. (e) Graphs show relative levels of pS133-CREB and pT410/403-aPKC over total CREB and aPKC, respectively, normalized to one of 3 months WT samples. (f) Western blot analysis for pS133-CREB in hippocampal extracts from 3 and 6 months C57B6 mouse strain. Blots were reprobed for total CREB as a loading control. Graphs show relative levels of pS133-CREB over total CREB, normalized to one of 3 months C57B6 samples. (g) Schematic model showing that both pS133-CREB and pS422-p300 determine the interaction between p300 and CREB. (h) Co-immunoprecipitation analysis of the association of p300 with pS133-CREB and total aPKC in the hippocampi of 6 months WT and p300G422S-KI mice. (i) The graph indicates the fold changes of the relative pulled-down pS133CREB protein and total aPKC over the total p300 amounts, as determined by densitometry and normalized to 6 months WT samples. exp1: individual experiment 1; exp2: individual experiment 2. *p < 0.05; **p < 0.01, n = 3–5 animals for each group. Images derived from different part of the same gel were cropped for layout reasons. Full-length blots/gels are presented in Supplementary Fig. 2.