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Fig. 5. The PIKfyve[S318A] mutant enhances insulin-stimulated IRAP translocation. (A) 3T3-L1 adipocytes stably expressing IRAP with an exofacial (luminal) HA tag, were microinjected with plasmids encoding GFP, wild-type GFP-PIKfyve (WT) or the PIKfyve[S318A] mutant (S318A). The cells were then treated in the absence (Con) or presence of insulin (Ins) as indicated. Cells were fixed, but not permeabilised, and stained with an anti-HA antibody that only detects surface-exposed IRAP. GFP-expressing cells exhibiting a clear surface-associated staining (i.e. a plasma membrane ring of fluorescence) were counted as having undergone IRAP translocation. In the absence of insulin, no cells were detected that exhibited surface staining. Quantitative data are provided for either two (control) or four (insulin) independent experiments. (B) Images of individual insulin-stimulated cells from one representative experiment are shown. HA-stained surface IRAP is shown in red, with the corresponding wild-type (WT, upper panel) or [S318A] mutant GFP-PIKfyve (lower panel) expression in the same cell in green. In this experiment 13 cells out of 33 wild-type GFP-PIKfyve expressing cells exhibited the translocation phenotype (39.3%). In cells expressing PIKfyve[S318A] this became 12 out of 19 (63%). Bar, 10 µm.
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