FIGURE 4.

AS160 is phosphorylated upon CA-Rac-GFP overexpression and is required for the Rac-1-induced gain in surface GLUT4. A and B, FLAG-AS160 was co-expressed with GFP, WT-Rac-GFP, or CA-Rac-GFP before immunoprecipitation (IP) with anti-FLAG antibody. Immunoprecipitates were stained with phosphorylated Akt substrate (PAS) antibody to detect changes in AS160 phosphorylation. The intensity of phosphorylated Akt substrate/total FLAG-AS160 in each condition was quantified and is depicted as the percentage of the parallel, maximal insulin response in units normalized to the GFP-expressing control. C and D, FLAG-AS160–4A was co-transfected with GFP or CA-Rac-GFP followed by stimulation with/without insulin (INS) to measure changes in surface GLUT4. Quantification of each condition is presented as the percentage of the parallel, maximal insulin response normalized to GFP-expressing insulin-stimulated cells (mean ± S.E. (error bars); *, p < 0.05). Representative images of three independent experiments are shown. Bar, 10 μm. IB, immunoblot.