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. 2017 Jun 28;7:4336. doi: 10.1038/s41598-017-04499-7

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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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FCCS and K_d analysis of mCherry₂-GRα and EGFP-GRα. Typical auto- and cross-correlation curves constructed by measurements in U2OS cells coexpressing the pairs of chimeric fusion proteins before and after addition of the ligand. The filled green diamonds, red squares, and gray triangles denote the autocorrelation of the green channel [G _G(τ)], autocorrelation of the red channel [G _R(τ)], and the cross-correlation curve [G _C(τ)], respectively, with their fits (solid black line) and residuals. The insets show LSM images of U2OS cells coexpressing the pairs of chimeric fusion proteins. Measurement positions of FCCS are indicated by the white crosshairs. The scale bars are 10 μm. FCCS was performed in U2OS cells expressing mCherry₂ and EGFP as a negative control (a) before and (b) after addition of Dex, showing a flat cross-correlation amplitude. (c) A U2OS cell coexpressing p50-mCherry₂/NLS and p50-EGFP/NLS as a positive control. (d) A U2OS cell coexpressing mCherry₂/hGRα and EGFP/hGRα in the cytoplasm before addition of Dex. (e) A U2OS cell coexpressing mCherry₂ /hGRα and EGFP/hGRα in the nucleus 20 min after addition of 100 nM Dex. (f,g,h) Results of K_d determination using a scatter plot and linear regression. The plots represent the square of the concentration of the monomeric hGRα versus the concentration of the dimer of hGRα. The solid lines show the linear fit. The slope indicates K_d. (f) mCherry₂-hGRα and EGFP-hGRα before addition of Dex. (g) mCherry₂-hGRα and EGFP-hGRα after addition of Dex. (h) p50-mCherry₂/NLS and p50-EGFP/NLS.