Figure 1

Spectrofluorimetric analysis of purified ShadowG.

(a) Purified proteins (300 μM) under daylight (top) and blue LED light (bottom) are shown. (b) Normalized absorption spectra of mEGFP, sREACh and ShadowG. (c) The emission spectra of mEGFP, sREACh and ShadowG excited at 470 nm. For all samples, optical density at 470 nm is adjusted to the same value. (d) An enlarged view of panel c. (e) The fluorescence lifetime curves of the fluorescent proteins. 920 nm was used for 2-photon excitation. (f) Curves of refolding of sREACh and ShadowG from a denatured state. Three independent experiments were performed (the data are shown as mean ± SEM). (g) Fluorescence recovery of sREACh and ShadowG from a denatured/reduced state. Three independent experiments were performed (the data are shown as mean ± SEM). (h) A fluorescence lifetime image of HEK293 cells expressing EGFP-CAAX (a motif of K-Ras) which localizes on the plasma membrane. The cells also express cytosolic mCherry, ShadowG, or sREACh. The fluorescent images were taken with the indicated band pass (BP) filters. Arrow heads indicate bleed-through fluorescence of sREACh. The scale bar is 50 μm. (i) Quantification of (h). The fluorescence lifetime of cells expressing EGFP-CAAX along with mCherry, ShadowG, or sREACh was compared. A fluorescence lifetime decay curve averaged over the whole image were analyzed. The number of images is 10 for all conditions. Each image contains 3–8 cells. The data are presented as mean ± SEM. Asterisks denote statistical significance (p < 0.05, analysis of variance [ANOVA] followed by Scheffé’s post hoc test).