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. 2018 Aug 22;8:12560. doi: 10.1038/s41598-018-29685-z

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© The Author(s) 2018

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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Biochemical and spectroscopic characterization of SERCA2a-PLB biosensor. (a) Immunoblots of homogenates from untransfected HEK293 cells (lane 1), cells expressing GFP-SERCA2a (lanes 2-3), or cells expressing GFP-SERCA2a and increasing amounts of RFP-PLB (lanes 4–8). Antibodies from top to bottom are anti-GFP, anti-SERCA2, anti-RFP, anti-PLB and anti-β-actin, and the image has been cropped. (b) Confocal fluorescence imaging of HEK293 cells expressing GFP-SERCA2a (left) or GFP-SERCA2a and RFP-PLB (right). (c) FRET efficiency E from GFP-SERCA2a (donor) to RFP-PLB (acceptor) shows hyperbolic dependence on acceptor concentration. (d) The mean FLT in the presence of saturating acceptor is τ_DA = 2.35 ± 0.02 ns, corresponding to E = 0.103 ± 0.004. Error bars indicate SD (n = 3). Statistical differences determined using the Student’s t test: *P ≤ 0.01.