Figure 4.
Characterization of GLUT4 accumulation in the evanescent field and fraction of this GLUT4 inserted into the PM. (A) Schematic of the method to measure HA-GLUT4-GFP accumulation in the evanescent field (GFPTIRF/GFPEPI fluorescence ratio) and the fraction of GLUT4 in the evanescent field that is inserted into the PM (αHA Cy3TIRF/GFPTIRF fluorescence ratio). (B–D) Characteristics of HA-GLUT4-GFP translocation (αHA Cy3EPI/GFPEPI fluorescence ratio), the insertion efficiency (fraction of GLUT4 in the evanescent field that is inserted into the PM; αHA Cy3TIRF/GFPTIRF fluorescence ratio) and accumulation in evanescent field (GFPTIRF/GFPEPI fluorescence ratio) in unstimulated and insulin-stimulated adipocytes. Cells were treated with DMSO, 1 μM Akti1/2, or 100 nM wortmannin 30 min before and during the 30-min insulin stimulation. Data are averages ± SEM from six independent experiments normalized to the control in the basal state. *p < 0.05, **p < 0.001 compared with the control in the insulin-stimulated state; #p < 0.05.