FIG. 7.

Overexpression of APS blocks insulin-stimulated GLUT4 translocation to the plasma membrane. (A) 3T3-L1 adipocytes were electroporated with 100 μg of GLUT4-EGFP plus 300 μg of vector, Myc-tagged wild-type APS (Myc-APS/WT), or Myc-APS/Y⁶¹⁸F and allowed to recover for 30 h. The cells were treated with or without 100 nM insulin for 30 min. Cells were fixed, and fluorescence was visualized by confocal microscopy. GLUT4-EGFP was visualized by direct fluorescence, and Myc-APS was visualized by indirect immunofluorescence. These are representative images of middle sections of cells obtained from three independent experiments. (B) Numbers of GLUT4-EGFP-transfected cells displaying visually detectable plasma membrane (PM) rim fluorescence. These data were obtained by blind counting of more than 80 cells from three independent experiments. Error bars indicate standard deviations. (C) 3T3-L1 adipocytes were electroporated with 100 μg of GLUT4-EGFP plus 100 μg of Myc-APS/Y⁶¹⁸F. After treatment with 100 nM insulin for 30 min, cells were fixed and fluorescence was visualized by confocal microscopy as described for panel A. This is a representative field of a middle section of cells obtained from three independent experiments.