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. 2019 Apr 30;9:6705. doi: 10.1038/s41598-019-42857-9

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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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Effect of PKA and cAMP on the expression of GRTH wild type and mutants that caused reduction or abolition of pGRTH at T239. (A and B) Western Blot analyses of GRTH and mutants co-expressed in COS-1 cells with or without PKA-Cα construct or empty vector. Western Blots of cytoplasmic cell fraction were probed with site-specific GRTH phospho-antibody and PKA-Cα antibody. (C and D) Western blot of the cytoplasmic fraction prepared from cells transfected with GRTH wild type and mutants and incubated with or without 500 mM cAMP for 24 h were probed with site-specific GRTH phospho-antibody. Expression of phospho-CREB was shown as positive control indicating cAMP activity. The protein band intensities of pGRTH from three independent experiments (mean ± SEM), measured using ImageJ software were normalized by β-actin. ¥ indicates significant difference between with or without PKA and cAMP groups. * and # indicates significant reduction in the expression of pGRTH compared to WT group (P < 0.05, One-way ANOVA with Tukey’s multiple comparison test). Full-length blots are presented in Supplementary Figs S5 and S6.