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. 2017 Nov 29;7:16552. doi: 10.1038/s41598-017-16457-4

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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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Palmitoylation-deficient mutant S1P₁R(3CA) was unable to couple with Gi. (a) SH-SY5Y cells transiently expressing wild-type S1P₁R-FLAG or S1P₁R(3CA)-FLAG were analysed for palmitoylation by ABE assay. One of the representative results from three independent experiments is shown. Full-length images are presented in Supplementary Figure 9. (b) SH-SY5Y cells transiently expressing S1P₁R(3CA)-CFP were stimulated with or without 100 nM S1P for 30 min and fixed with paraformaldehyde. The localisation of S1P₁R(3CA)-CFP was analysed by confocal microscopy. Bars, 10 µm. One of the representative results from three independent experiments is shown. (c) SH-SY5Y cells transiently expressing S1P₁R(3CA)-CFP were stimulated with 100 nM S1P for various time periods as specified. After fixation of the cells, the fluorescence of each protein was analysed by confocal microscopy. Fluorescence intensity at each time point was quantitated and compared with time point 0 (control). Values represent means ± s.e.m. of 3 independent experiments carried out in triplicate. Statistical significance was analysed by Student’s t-test (n = 9; **P < 0.01, *P < 0.05 versus control (0 min)). (d) SH-SY5Y cells transiently expressing wild-type S1P₁R-CFP or S1P₁R(3CA)-CFP together with Gβ and Gγ-YFP were stimulated with or without 100 nM S1P for 2 min, fixed and analysed for FRET efficiencies. Values represent means ± s.e.m. (n ≥ 50). Statistical significance was analysed by Student’s t-test (*P < 0.05 versus wild-type S1P₁R with S1P).