FIGURE 3.

Inhibition of GLUT4 translocation by TBC1D1 phosphorylation mutants. A, relative amounts of surface GLUT4 in basal and insulin-stimulated 3T3-L1 adipocytes transfected with HA-GLUT4-GFP plasmid, together with the control vector plasmid alone (V) or with the plasmids for wild-type (WT) and phosphorylation site mutants (1P, 2P, and 3P) of TBC1D1 (TBC). The values are the mean ± S.E. from two replicate experiments. Figs. 4A and 5A show six additional experiments in which TBC1D1 wild-type and 3P mutant were among those examined. The lower amount of GLUT4 at the cell surface in the insulin state for the 3P mutant compared with wild-type TBC1D1 for these eight experiments was significant, with p < 0.05. B, SDS lysates of cells in A were immunoblotted for TBC1D1 with anti-FLAG antibody. The 1× load was 10 μg. A representative immunoblot is shown.