Glut3 is localized at rab11-positive endosomes. A), Co-localization of Glut3 and rab11 in primary cortical neurons. Primary WT neurons on glass coverslips were processed for immunofluorescence labeling of endogenous Glut3 (red) and endogenous rab11 (green) analyzed by confocal microscopy. Yellow areas in the merged photo show signals positive for both Glut3 and rab11. The boxed region containing part of a neurite in each channel is enlarged and shown at the lower left corner of the corresponding channel. Arrowheads in enlarged photos indicate co-localization of Glut3 and rab11. B), Co-distribution of Glut3 with rab11 in subcellular brain fractions. Density gradient ultracentrifugation was conducted as described in Methods. Equal volume of each fraction was used for Western blot analysis. Shown is one blot analysis of three experiments from three mice. C), Glut3 is present in organelles immuno-isolated from brain lysates using anti-rab11 antibodies. Fraction-5 from (B) was diluted in detergent-free buffer and incubated with antibodies specific for rab11 or FLAG pre-immobilized on protein-A resins. After washes in detergent-free buffer, proteins on protein-A resins were analyzed by Western blot with antibodies against Glut3. Shown is a Western blot analysis from one of three experiments.