Figure 3.

(A) Fluorescence emission spectra of WCB-eENTH in response to binding to POPC/POPS/PI4,5P₂ (80–x/20/x: x = 0–2 mol%) LUVs. The spectra were obtained spectrofluorometrically with the excitation wavelength set at 380 nm. The black line represents the spectrum of the sensor alone and the red line that with 0% PI4,5P₂ (i.e., POPC/POPS (80/20)) (B) The ratiometric calibration curve of WCB-eENTH for PI4,5P₂ quantification. The calibration was performed as described for Figure 2B except that the blue channel represents the membrane-bound sensor. (C) Representative blue-channel and green channel cross-sectional images of NIH 3T3 cell containing WCB-eENTH. (D) A spatial PI45P₂ concentration profile calculated from Figure 3C. The z-scale indicates mol% of PI4,5P₂. The pseudo-coloring scheme is the same as in Figure 2D. (E) Relative photostability of WCB-eENTH and DAN-eENTH in NIH 3T3 cells. Total blue-channel fluorescence intensity at the PM of a fixed number (e.g., 15) of cells was counted as a function of time with continuous illumination. (F) Fluorescence emission spectra of WCR-eMyoX-tPH in response to binding to POPC/POPS/PIP₃ (80–x/20/x: x = 0–1 mol%) LUVs. The spectra were measured with the excitation wavelength set at 488 nm. The black line represents the spectrum of the sensor alone and the red line that with 0% PIP₃ (i.e., POPC/POPS (80/20)) (G) The ratiometric calibration curve of WCR-eMyoX-tPH for PIP₃ quantification. The calibration was performed as described for Figure 2B except that the ratio of fluorescence intensity in the orange channel to that in the red channel was used for analysis. (H) Representative orange- and red-channel cross-sectional images of NIH 3T3 cell containing WCR-eMyoX-tPH. (I) A spatial PIP₃ concentration profile calculated from Figure 3H. The z-scale indicates mol% of PIP₃. A pseudo-coloring scheme was used with red and blue representing the highest (i.e., 0.6 mol%) and the lowest (i.e., 0 mol%) concentration of PIP₃, respectively. (J) Relative photostability of WCR-eMyoX-tPH and NR3-eMyoX-tPH in NIH 3T3 cells. Total orange-channel fluorescence intensity at the PM of a fixed number (e.g., 15) of cells was counted as a function of time with continuous illumination. Scale bars indicate 5 μm.