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. 2017 Jul 5;7:4695. doi: 10.1038/s41598-017-04835-x

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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 pBMP-9 and SpBMP-9 on the PI3K/Akt/GSK3β pathway. (A) Merged pictures representative of at least 2 independent experiments showing immunostaining for pAkt (Thr308) (green) and nuclei (Hoechst, blue) of SH-SY5Y cells stimulated for 2 h with 0.1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA (Bar = 100 μm) and pAkt(Thr308) fluorescence activity relative to the nucleus. (B) Western blots of phosphorylated GSK3β at Ser9 (pGSK3β) and densitometric analysis of pGSK3β bands standardized by actin showing the effect of 0.1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA in SH-SY5Y cells after incubation for 0, 15, 30, 60, 120 and 240 min. Only cropped pictures of western blots were shown in order to allow a better comparison between experimental conditions. Complete gel pictures are available in the supplementary data file. (C) Merged pictures representative of at least 2 independent experiments showing immunostaining for pGSK3β (Ser9) (FITC, green) and nuclei (Hoechst, blue) in SH-SY5Y cells stimulated for 4 h with 0.1 nM BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA (Bar = 100 μm). Analysis of pGSK3β(Ser9) fluorescence intensity relative to the nucleus staining was also presented. Results are the means ± SEM (**p < 0.01, ***p < 0.001).