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. 2017 Aug 11;7:7885. doi: 10.1038/s41598-017-07392-5

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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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Gα expression level influences Gα subunit and β-AR receptor compartmentalization in cholesterol-enriched membranes. Detergent-resistant-membranes were purified using a triton X-100 lysis method followed by a separation on sucrose gradient from (a) HEK293T cells co-expressing high (orange) and low (black) expression levels of Gαs-Rluc8 (left panel), Gαi1-Rluc8 (middle panel) or GαoA-Rluc8 (right panel) along with Gβ3 and Gγ2 subunits in the presence or not (upper panels) of untagged β-AR receptors, (b) HEK293T cells co-expressing β1-AR-Rluc or β2-AR-Rluc alone (upper panels) or in the presence of high (orange) or low (black) expression levels of untagged Gαs (left panels), Gαi1 (middle panels) or GαoA (right panels) and Gβ3 and Gγ2 subunits. Relative receptor or Gα subunit expression levels were quantified in each sucrose fractions by recording of the total luminescence. Results are expressed as the percentage of the maximal luminescence measured from all fractions in each experiment. Grey boxes highlight raft nano-domains enriched fractions, identified by detection of the GM1 protein (upper dot plot). Data represent the mean ± s.e.m. of at least three independent experiments. The statistical significance of the difference in membrane distribution in low versus high conditions (*) or versus receptor alone (¤) was assessed using two-way ANOVA followed by a Bonferroni posttest (*P < 0.05, **P < 0.01, ***P < 0.001).