Fig. 5. Quantitative analysis of the dominant-negative effect of PKB[KD]-GLUT4 and Myr-PKB[KD]. In A and C, cells were scored for the visual presence of IRAP-GFP in the plasma membrane by double blind analysis by two independent workers as discussed in the Materials and Methods. The results shown represent means±s.d. for three independent experiments with each condition being represented by a minimum of 50 cells. ^*P<0.01 versus the vector only injected cells incubated in the presence of insulin. In B the amount of IRAP-GFP residing in the plasma membrane was calculated as a percentage of the total expressed IRAP-GFP in that cell for each condition. The results are displayed as means±s.d. for a minimum of 10 cells for each condition, and they were collected from at least three separate experiments. ^*No significant difference from the control without insulin incubation. In C, adipocytes were microinjected with plasmids encoding IRAP-GFP and either kinase-inactive PKB (PKB[KD]), kinase-inactive PKB lacking the PH domain (PH-PKB[KD]) or kinase-inactive PKB fused to the N-terminus of GLUT4 (PKB[KD]-GLUT4). 16 hours later the cells were serum starved for 2 hours and then incubated in the absence or presence of insulin for 30 minutes. The cells were then scored for the presence of IRAP-GFP in the plasma membrane as discussed in the Materials and Methods. The results shown represent means±s.d. for three independent experiments, with each condition being represented by a minimum of 50 cells. ^*P<0.01 versus the PKB[KD]-injected cells incubated in the presence of insulin.

Return to article