FIGURE 4.

AS160(1–532) interacts with full-length AS160 and reduces the relative amount of 14-3-3γ bound to AS160 and associated with GLUT4 vesicles. A, primary rat adipocytes were cotransfected by electroporation with HA-tagged GLUT4 and FLAG-tagged AS160(1–532). After 5 h of expression, cells were maintained in the basal state (B) or stimulated with 60 nm insulin (I). HA-GLUT4 present at the cell surface was detected after incubating the intact cells with anti-HA antibody for 1 h and detection using a β-galactosidase-coupled secondary antibody generating a fluorescent product. Results are means ± S.E. from four replicates from a single experiment representative of two independent experiments. *, p < 0.05 (comparison of HA-GLUT4 alone versus HA-GLUT4 plus FLAG-AS160(1–532)). B–D, primary rat adipocytes were cotransfected by electroporation with FLAG-tagged full-length AS160 and HA-tagged AS160(1–532). After 12 h of expression, cells were left unstimulated or stimulated with 60 nm insulin and then homogenized. GLUT4 vesicles (G4V) were immunoisolated, and the relative amounts of 14-3-3γ (B), AS160 (C), and phosphorylated AS160 (pAS160; D) were detected by immunoblotting with anti-14-3-3γ, anti-AS160, and anti-PAS antibodies, respectively. Results are means ± S.E. from three independent experiments. E, representative immunoblots for data quantified in B–D. *, p < 0.05 (comparison of FLAG-AS160 alone versus FLAG-AS160 plus HA-AS160(1–532)).