Figure 10.

Pixel-by-pixel quantification of sensitized emission FRET between mGAT1⁵⁶⁵XFP⁵⁶⁶CT and mGAT1⁵⁷⁰XFP⁵⁷¹CT. (A; from left to right) mGAT1⁵⁶⁵CFP⁵⁶⁶CT fluorescence and mGAT1⁵⁶⁵YFP⁵⁶⁶CT fluorescence (calibration bars, ACUs), ROIs used to determine NFRET (color coding as in Fig. 8), and the NFRET image (color calibration bar, NFRET × 100) of the same cell. Pixels with signal amplitude below threshold are shaded gray. Bars, 10 µm. (B) Box plots displaying NFRET for all pixels in each ROI of cells expressing mGAT1⁵⁶⁵XFP⁵⁶⁶CT. The box, whiskers, and other data points are represented as in Fig. 7 B. Box plots for each ROI are colored according to the code in Fig. 8 A (ROI), and the intracellular data are colored blue. (C) Table of results for mGAT1⁵⁶⁵XFP⁵⁶⁶CT transfections displaying the mean of each Gaussian component of the summed fit of the distributions in D and the percentage of the pixels comprising each component. (D) Distributions of NFRET signal amplitude per pixel for each ROI (bin width, 0.02) from mGAT1⁵⁶⁵XFP⁵⁶⁶CT-expressing cells. The individual components and the sum of the fit are shown as dashed and solid lines, respectively. (E) Representative fluorescence images of mGAT1⁵⁷⁰XFP⁵⁷¹CT-expressing N2a cells, the regions of interest, and corresponding NFRET image. Calibration and color coding are as for Fig. 8 A. (F) Box plots displaying NFRET for all pixels in each ROI of cells expressing mGAT1⁵⁷⁰XFP⁵⁷¹CT. Color coding and data are represented as in B. (G) Table of results for mGAT1⁵⁷⁰XFP⁵⁷¹CT transfections displaying the mean of each Gaussian component of the summed fit of the distributions in H and the percentage of the pixels that comprise each component. (H) Distributions of NFRET signal amplitude per pixel for each ROI (bin width, 0.02) from mGAT1⁵⁷⁰XFP⁵⁷¹CT-expressing cells. The individual components and their sum are shown as dashed and solid lines, respectively.