Fig. 2. Phosphorylation of HA-GLUT4-Akt2 fusion proteins at Ser⁴⁷⁴ and Thr³⁰⁹ in rat adipose cells. Isolated cells were transfected with HA-GLUT4-Akt2-wt (WT) and HA-GLUT4-Akt2-K179A (KD), and cultured for 20 hours. After harvesting the cells, total cell membrane proteins were prepared from the transfected cells in the basal and insulin-stimulated states (67 nM) by differential centrifugation. 40 µg of protein were subjected to immunoblotting with anti-Akt-phospho-Ser⁴⁷³ or -phospho-Thr³⁰⁸ antibody. These data are representative of 3 independent experiments.