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. 2023 Sep 12;13:15080. doi: 10.1038/s41598-023-41166-6

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Phosphorylation levels of AKT, mTOR, and FOXO3a after 24 h of treatment with insulin 100 nM or Klotho 1 nM in primary neuronal culture. (A) Statistical analysis indicated that insulin increases AKT phosphorylation on Ser473, while Klotho decreased AKT phosphorylation. One-way ANOVA followed by Tukey’s multiple comparisons tests; pAKT/AKT [F = 29.05; P = 0.0001; R squared: 0.8659]. (B) Statistical analysis of mTOR phosphorylation levels. One-way ANOVA followed by Tukey’s multiple comparisons tests indicated that insulin increased phosphor-mTOR, as expected, whereas Klotho decreased phospho-mTOR[F = 25.78; P = 0.0002; R squared = 0.8514]. (C) Statistical analysis of FOXO-3a phosphorylation levels. One-way ANOVA indicated the absence of significant difference [F = 0.8042; R squared = 0.1182, P = 0.4702]. Below the graphs are the representative bands of each group in western blot autoradiographs. The original autoradiographs are available in Supplementary Fig. S3. N = 4–5 independent cell cultures. *P < 0.05 and ***P = 0.0001.