Figure 7.

The effects of VAMP knockdowns on GLUT4 endocytosis. Fully differentiated adipocytes were cotransfected with a random siRNA or VAMP-specific siRNA plus the myc-GLUT4-GFP cDNA reporter. 72 h later, the cells were stimulated with 100 nM insulin for 30 min and cooled to 4°C. The exofacial exposed myc epitope was labeled with a myc antibody, and the cells were washed to remove insulin and excess antibody and subsequently were warmed to 37°C. At various times, the cells were fixed, permeabilized, and incubated with an AlexaFluor594 anti–mouse secondary antibody, and the cells were scored for the presence of internalized GLUT4 as described in Materials and methods. These data represent the analysis of 15 cells/experiment from three independent experiments. Error bars represent SD.