Fig. 5. Basal and insulin-stimulated cell-surface distributions of wild-type HA-GLUT4 and C-terminal deletion mutants in rat adipose cells. Isolated cells were transfected with 1.25 µg/ml plasmid DNA and cultured for 4 hours. In the case of the 37 mutant, 12.5 µg/ml plasmid DNA were used. After harvesting, the cells were incubated without (basal, solid bars) or with (open bars) 67 nM insulin for 30 minutes, and the cell-surface levels of HA-GLUT4 were determined using an antibody binding assay as described in Materials and Methods. The cell surface-associated radioactivity was normalized to the relative protein expression level of each respective mutant (Fig. 2). Results are the means±s.e.m. of at least three replicate experiments performed in duplicates to quadruplicates.