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. 2020 Apr 15;11:1848. doi: 10.1038/s41467-020-15687-x

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© The Author(s) 2020, corrected publication 2024

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. 2 — a stagRFP confers a significant increase in FRET dynamic range. The mean ± SEM is displayed. Stimulation of PKA in HeLa or HEK293T cells was performed using 50 μM forskolin and 100 μM IBMX, while inhibition was performed using 20 μM H89. *p < 0.05; **p < 0.01; ***p < 0.0001 from two tailed, unpaired Welch’s t-test. Specifically, p values for the comparison between stagRFP and mScarlet in the AKARev context p = 0.0225 (t = 2.349, df = 55); between stagRFP and mCherry in the mClover-AKARev context p = 0.0089 (t = 3.239, df = 10); between stagRFP and mCherry in the EGFP-AKAR context p = 0.0430 (t = 2.080, df = 47); and between stagRFP and mRuby in the AKAR context p < 0.0001 (t = 15.80, df = 47). The changes in normalized emission ratio (red/green) are AKAR2-CR, 8.1 ± 0.8%, n = 32 biologically independent cells; EGFP-mCherry-AKAR, 17.2 ± 0.8%, n = 38; EGFP-stagRFP-AKAR, 19.5 ± 0.7%, n = 17; mClover-stagRFP-AKAR, 21.9 ± 1.7%, n = 35; mClover-mCherry-AKARev, 27.5 ± 2.9%, n = 6; EGFP-mScarlet-AKARev, 38 ± 1.8%, n = 21; mClover-stagRFP-AKARev, 39.1 ± 2.0%, n = 35; EGFP-stagRFP-AKARev, 44.6 ± 2.1%, n = 37. b Diffusible GR-AKAR3 displays a high dynamic range (n = 8). The mean ratio ± SEM is displayed. Source data are provided as a Source Data file. c Membrane-targeted biosensor is able to detect the increase in membrane PKA activity (n = 20). The mean ratio ± SEM is displayed. d The change in mean donor lifetime normalized to initial donor lifetime of GR-AKAR3 in HeLa cells upon Fsk/IBMX stimulation (n = 6). The mean ratio ± SEM is displayed in all panels.