Figure 6
From: ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
The performance of ShadowY in H-Ras FRET sensors in HeLa cells. (a) A schematic of the H-Ras FRET sensor activation. (b) Representative fluorescence lifetime images of mEGFP-H-Ras paired with ShadowY fused to the Ras-binding domain (RBD) in HeLa cells after stimulation with 50 nM EGF. The scale bar is 50 µm. (c) An averaged time course of binding fraction changes in response to EGF stimulation (filled circles) or with empty buffer (open circles; control). Cells showing a 5–30% basal binding fraction (before stimulation) were chosen for the analysis. The number of cells analyzed is 61 for mEGFP–ShadowY, 70 for Clover–ShadowY, and 61 for CloverT153M/F223R–ShadowY. In the control, the number of cells analyzed is 45 for mEGFP–ShadowY, 18 for Clover–ShadowY, and 31 for CloverT153M/F223R–ShadowY. The data are presented as mean ± SEM. (d) Binding fraction changes (averaged over 8 to 10 min) after EGF stimulation. The data are presented as mean ± SEM; N. S. = not statistically significant (p > 0.05, analysis of variance [ANOVA] followed by Scheffé’s post hoc test). (e–g) Activation of H-Ras in individual HeLa cells after stimulation with EGF (the same dataset as in panel c). The basal binding fraction (averaged over −2 to 0 min) of individual cells is plotted in the descending order (black) along with the corresponding binding fraction (averaged over 8 to 10 min) after EGF stimulation (red). The data are also presented as mean ± SD on the right.
