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

Figure 6.

Figure 6

The effect of NLS mutation on formation of the GRα dimer. Typical auto- and cross-correlation curves obtained from 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 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 lines) and residuals. The insets show LSM images of the U2OS cells coexpressing the pairs of chimeric fusion proteins. FCCS analyses were carried out in the cytoplasm, which is indicated by the white crosshairs. The scale bars are 10 μm. FCCS was performed using U2OS cells coexpressing (a) p50-mCherry2 and p50-EGFP as a positive control, (b) mCherry2-hGRα/∆NLS and EGFP-hGRα/∆NLS before addition of Dex, (c) mCherry2-hGRα/∆NLS and EGFP-hGRα/∆NLS 20 min after addition of 100 nM Dex, or (d) mCherry2-hGRα/A458T-∆NLS and EGFP-hGRα /A458T-∆NLS 20 min after addition of 100 nM Dex. (e–h) Results of Kd determination using scatter plots and linear regression. The plots represent the square of the concentration of the monomeric hGRα versus the concentrations of hGRα dimer. The solid line shows the linear fit. The slope indicates the Kd. (e) p50-mCherry2 and p50-EGFP. (f) mCherry2-hGRα/∆NLS and EGFP-hGRα/∆NLS before addition of Dex. (g) mCherry2-hGRα/∆NLS and EGFP-hGRα/∆NLS after addition of Dex. (h) mCherry2-hGRα/A458T-∆NLS and EGFP-hGRα/A458T-∆NLS after addition of Dex.