Fig. 1. Subcellular localisation of PKB mutants in 3T3-L1 adipocytes. 3T3-L1 adipocytes were microinjected with plasmids encoding wild-type PKB (A; PKB[WT]), kinase-inactive PKB (B; PKB[KD]), constitutively active PKB (C; PKB[DD]), constitutively active myristoylated PKB (D; Myr-PKB), constitutively active PKB fused to the N-terminus of GLUT4 (E,F; PKB[DD]G4) and kinase-inactive PKB fused to the N-terminus of GLUT4 (G,H; PKB[KD]G4). 16 hours later the cells were serum starved for 2 hours and then incubated in the absence (A-E,G) or presence (F,H) of transferrin-Alexa568 for 30 minutes (as indicated by `Tf-Alexa') and then fixed and stained with anti-HA antibodies to visualise the PKB construct (as indicated in the panel) and anti-IRAP antibodies (as indicated by `IRAP'), as required. A merged image is also provided of the regions indicated by the yellow box. Bars, 10 µm. The schematic of the PKB-GLUT4 fusion proteins illustrate the position of the N-terminal HA-tag (red), PKB (blue), GLUT4 (cyan) and the activatory (T308D/S473D) and inhibitory (K179A) mutations.

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