(A and B). Optimization of the dose of MK2206 and insulin to study insulin stimulated Glut4 translocation in 3T3-L1 adipocytes. Surface (Cy3 anti-HA) to total (GFP) ratio of HA-GLUT4-GFP is plotted in control adipocytes or adipocytes co-expressing Akt-W80A. Cells were pretreated with the indicated dose of MK2206 for 1 hr followed by 30 min stimulation by insulin. More than 30 cells were quantified per condition per assay. Mean normalized values ± SEM. n = 3–4 independent experiments. In panel A, the data of the individual experiments are normalized to Glut4 surface to total value in control cells stimulated with 1 nM insulin. In panel B, the data are normalized to Glut4 surface to total value in Akt2-W80A + MK2206, 0.2 nM insulin condition. (C and E). In a similar experimental setup, cell lysates were collected and subjected to immunoblot analysis for Akt and AS160 phosphorylation. Arrow heads note the migration of ectopically expressed Akt, whose migration is slower due to the amino-terminal Flag epitope. (D and F). The quantification for (C) and (E) respectively. (i-iii) control adipocytes, (iv-vi) adipocytes transiently transfected with ectopic Akt. Each data normalized to 1 nM insulin. n = 5 independent experiments.