FIG. 7.

Effect of insulin on the intracellular distribution of GLUT4 in 3T3-L1 adipocytes. (A) 3T3-L1 adipocytes were serum starved for 16 h in DMEM, incubated with or without 10⁻⁷ M insulin for 20 min, and then subjected to Tf-HRP ablation as described in the legend for Fig. 5 by using 25 μg of Tf-HRP conjugate per ml and 50 μg of DAB per ml in the presence (+) or absence (−) of 0.02% H₂O₂. The insulin level was maintained throughout the incubation with the Tf-HRP conjugate. The LDM fraction was obtained as described above and analyzed by SDS-PAGE followed by immunoblotting. Syn 6, syntaxin6; Ceb, cellubrevin. (B) 3T3-L1 adipocytes were serum starved for 16 h in DMEM at 37°C and then left untreated or stimulated with 10⁻⁷ M insulin for 20 min. The LDM fraction was obtained and subjected to iodixanol gradient sedimentation as described. The iodixanol fractions were analyzed as described in the legend to Fig. 3 by SDS-PAGE followed by immunoblotting for GLUT4. The results shown are representative of three separate experiments. Numbers represent different fractions.