Figure 3.

PI3K activity is required to promote Glut1 cell surface localization. FLAG-Glut1–expressing cells were cultured in the presence or absence of IL3 for 8 h with a vehicle control or 10 μM LY294002. (A) Cells were stained with a FLAG antibody, and mean surface levels were analyzed with flow cytometry. (B) Total FLAG expression was measured by immunoblot and Bcl-2 expression was measured for loading control. (C) FLAG-Glut1 cells were cultured in the absence of IL3 for 3 h. Vehicle control, 10 μM LY294002, and 10 μg/ml CHX were added 1 h before readdition of IL3. After an additional hour, cells were stained with FLAG antibody, and surface levels of FLAG-Glut1 were compared before and after IL3 add-back by flow cytometry. The differences in mean fluorescence of surface FLAG-Glut1 before and after IL3 readdition are shown. Mean fluorescence and SD of triplicate samples are shown. Asterisk (*) indicates p value <0.05 within the experiment. Representative results are shown for three or more repeated experiments.