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. 2019 Mar 4;2:88. doi: 10.1038/s42003-019-0338-1

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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. 4 — TRPV1 is in close proximity to Orai1. a Representative confocal images illustrating the expression of Orai1-YFP and TRPV1-CFP before (pre-bleach) or after (post-bleach) photobleaching of the acceptor (YFP). The square shows the area photobleached and the circle a control area not exposed to the photobleach procedure. We used both areas to calculate the FRET efficiency as indicated in Methods. Right panel shows the FRET efficiency (FRET eff) in pseudo color calculated from the acceptor photobleaching protocol (Methods). Scale bar 1 µM. b Representative confocal images illustrating the expression of Orai1-YFP and P2X₄-CFP before (pre-bleach) or after (post-bleach) photobleaching of YFP. The square shows the area photobleached and the circle a control area not exposed to the photobleach procedure. FRET efficiency (FRET eff) in pseudo color calculated from the acceptor photobleaching protocol (Methods). Scale bar 1 µM. c FRET eff (%) obtained from at least 45 cells for P2X₄-Orai1 and TRPV1-Orai1. Data show the mean ± standard deviation. d Co-immunoprecipitation analysis of cells expressing c-Myc-Orai1 and TRPV1-YFP or P2X₄-CFP (for the entire blots check Supplementary Figures). Total indicates the protein that was not retained in the affinity column, thus reflects the protein that did not co-immunoprecipitate (Methods). Blots are representative examples from at least 3 independent blots