Fig. 3. Effects of dominant negative dynamin on cell-surface expression (A) and Ser⁴⁷⁴ phosphorylation (B) of HA-GLUT4-Akt2-KD in rat adipose cells. Isolated cells were transfected with HA-GLUT4-Akt2-KD alone or co-transfected with HA-GLUT4-Akt2-KD and dominant negative dynamin, and cultured for 20 hours. After harvesting the cells, cell-surface levels of HA-GLUT4-Akt2-KD (A), and Ser⁴⁷⁴ phosphorylation of Akt2 in membrane fractions (B) were detected in the transfected cells in the basal (white) and insulin-stimulated states (67 nM, black) using an anti-HA antibody binding assay and immunoblotting, respectively. Results in A are the means ± s.e.m. of the means obtained from at least duplicate determinations in 3 independent experiments; results in B are representative of 2 independent experiments.