Figure 3.

Colocalization of the GLUT4 and Tf in the evanescent field. (A) Distribution of the HA-GLUT4-GFP and Tf-Alexa⁵⁴⁶ in TIRFM of basal adipocytes. Data were processed as discussed in Figure 2. Scale bar, 10 μm. (B) Colocalization of GLUT4 and Tf from an individual experiment. The percent colocalization is the percent of Tf vesicles (ROI identified in red channel) whose intensity in the green channel is above the green channel low-intensity threshold. Each symbol is the colocalization determined in an individual cell within the experiment. The lines are the average values. All the data were collected during one block of time on the microscopy; all the green channel images were processed with the same green channel low-intensity threshold, and all the red images were processed with the same red channel low-intensity threshold. Four to20 cells for each construct were analyzed in each individual experiment. (C) The average colocalization (percent of Tf vesicles positive for GLUT4) measured in four to five independent experiments ± SEM. The data illustrate the reproducibility of the measurements across independent experiments. (D and E) Colocalization of GLUT4 mutants with Tf normalized to WT GLUT4 colocalization with Tf. The colocalizations of the different mutants with Tf determined in individual experiments were normalized to the colocalization of WT GLUT4 measured in the same experiment, illustrating how the mutations affect the WT GLUT4 colocalization. (D) The percent of Tf vesicles (ROI identified in the red channel) that are also positive for GLUT4 are plotted. (E) The percent of GLUT4 vesicles (ROI identified in the green channel) that are also positive for Tf are plotted. The data are averages ± SEM of four to five independent experiments. The p values for paired t test are shown and are the comparison to WT GLUT4.