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. 2019 Jul 18;10:3187. doi: 10.1038/s41467-019-11227-4

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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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Fig. 8 — PKB/Akt activation is required for HDAC4 nuclear import and promotes endplate remodeling. a Changes in mTORC1 and PKB/Akt pathways in control (Ctrl), TSCmKO and Akt1-TG denervated muscles. b Western blot analysis of total and phosphorylated levels of endogenous and transgenic (Akt1-GFP) PKB/Akt, and of S6 in TA innervated and 3-day-denervated control, Akt1-TG (Akt1) and TSCmKO (TSC) muscles. Mice were treated with tamoxifen the day prior denervation and over the experiment period. n = 3 Ctrl, 3 Akt1-TG and 2 TSCmKO mice. c HE coloration (top panel) and fluorescent pictures of MHCIIA/X (bright/dark green) and laminin (red) immunostaining (bottom panel) of TA denervated (14d) muscle from control and Akt1-TG mice. Representative of 3 Ctrl, 3 Akt1-TG and 4 TSCmKO mice. Scale bar, 100 µm. d Immunofluorescent pictures of TA denervated (3d) muscle (6 independent muscles) from control and Akt1-TG mice, showing HDAC4 (red) and laminin (green). Arrows point to HDAC4-positive myonuclei. Scale bar, 100 µm. e Western blot analysis of total and phosphorylated HDAC4 in TA innervated (In) and denervated (De; 3d) muscles from control and Akt1-TG mice. n = 3 per group. f Proportion of HDAC4-positive myonuclei in TA denervated (3d) muscle from control and Akt1-TG mice. n = 3 per group. g, h Transcript levels of Eno2, Pfkm, Myh4, Mitr and Dach2 (g) and Myog and Chrng (h) in TA innervated and denervated muscles, from control and Akt1-TG mice. Transcript levels were normalized to Tbp mRNA and to Ctrl innervated muscle, and analyzed as the log2 of the fold change. n = 3 per group. i, j AChR turnover quantification in control and Akt1-TG innervated muscles, and 14 days after denervation (i). n = 3 per group. Representative pictures of “old” (green) and “new” (red) AChRs are given in (j). Arrows point to internalized AChRs. Scale bar, 50 µm. All values are mean ± s.e.m.; two-way ANOVA with Tukey’s post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Western blot quantifications are shown in Supplementary Table 1. Source Data are provided in the Source Data File