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Figure 4


Fig. 4. A dominant-interfering mutant form of AS160 (AS160-4P) inhibits the insulin-stimulated exit of IRAP-TfR-AA76,77 from the IRC following endocytosis from the cell surface. Fully differentiated 3T3L1 adipocytes were electroporated with 50 µg of IRAP-TfR-AA76,77 and either AS160-WT (white bars), or AS160-4P (black bars). Following a 16-hour recovery period, cells were surface-labeled with the anti-TfR antibody on ice for 60 minutes. Cells were then warmed to 37°C for 6 hours to allow endocytosis to occur, then treated without or with 100 nM insulin for 30 minutes, fixed, permeabilized and labeled with Texas-Red-conjugated secondary antibody as described under Materials and Methods. The ratio of plasma membrane fluorescence to total fluorescence was determined using the Zeiss LSM software package (mean ± s.e.m. of three independent experiments). a.u., arbitrary units.





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