Fig. 6. Clearance of insulin-stimulated cell-surface HA-GLUT4 and HA-GLUT4 mutants in rat adipose cells. Isolated cells were transfected with 1.25 µg/ml plasmid DNA (except for 37 where 12.5 µg/ml plasmid DNA were used) and cultured for 4 hours. After harvesting, the cells were stimulated with 67 nM insulin for 30 minutes. Wortmannin (100 nM) was then added at time 0 and the cell-surface levels of HA-GLUT4 were determined at the indicated time points using an antibody binding assay as described in Materials and Methods. , HA-GLUT4 wild-type; , F5A; , 37. Results are the means±s.d. of 2-4 replicate experiments performed in duplicates to quadruplicates.