Rab11 regulates Glut3 trafficking. A) Defective glucose uptake in immortalized STHdhQ111/Q111 striatal cells. [3H]deoxyglucose uptake was performed in PBS at room temperature for 20 minutes. After uptake, cells were washed three times in cold PBS and cell lysates were prepared. The amount of [3H]deoxyglucose in cell lysates was measured by liquid scintillation. Mean ± SD cpm per minute per milligram protein was calculated and graphed (N = 3, Two tailed Student t-test). B) Western blot analysis of post-nuclear supernatants showed up-regulated Glut3 protein expression in STHdhQ111/Q111 cells relative to STHdhQ7/Q7 cells. C) STHdhQ7/Q7 cells were cultured on glass coverslips, fixed and processed for immunofluorescence labeling of endogenous Glut3 (green), endogenous rab11 (red), and nuclei (blue). The boxed region is enlarged and shown at the lower left corner of the corresponding channel. Arrows in enlarged photos indicate structures containing both Glut3 and rab11. D) Western blot analysis of Glut3 levels upon expression of rab11 mutants. STHdhQ7/Q7 cells in 60-mm dishes were infected with lentivirus expressing dNrab11, or virus expressing dArab11, or uninfected (No virus) for two days. Biotinylated proteins and corresponding total cell lysates were analyzed by Western blot. The results from one of three experiments are shown. E) Densitometry of biotinylated Glut3 and corresponding background signals in each condition using NIH ImageJ. The ratio of biotinylated Glut3 signal from cells infected with virus to biotinylated Glut3 signal from cells with no virus infection was graphed (n = 3 experiments, Mean ± SD, Student t-test: *p < 0.05; **p < 0.01). F) Effects of dArab11 or dNrab11 on cell surface expression of Glut3 in STHdhQ111/Q111 cells. Experimental procedures and data analysis were performed as in D). Shown are blot analysis from one of two experiments.