Fig. 4. Cell-surface expression (A) and Ser⁴⁷⁴-phosphorylation (B) of HA-GLUT4-Akt2-S474A and -T309A constructs in rat adipose cells. Isolated cells were transfected with HA-GLUT4, HA-GLUT4-Akt2-wt, HA-GLUT4-Akt2-S474A, and HA-GLUT4-Akt2-T309A, and cultured for 20 hours. After harvesting the cells, cell-surface levels of the constructs were determined in the transfected cells in the basal (white) and insulin-stimulated states (67 nM, black) using an HA antibody binding assay and the cell-surface-associated radioactivity was expressed relative to the basal HA-GLUT4 value in each experiment (1668, 1558, 2311 cpm). Relative cell-surface levels were then normalized to the total expression level of each construct determined by immunoblotting as described in Materials and Methods. Results are the means ± s.e.m. of the means obtained from at least duplicate determinations in each of 3 independent experiments (A). Results in B are immunoblotting of total membrane proteins prepared from the transfected cells with anti-Akt-phospho-Ser⁴⁷³ antibody. These data are representative of 3 independent experiments.