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. 2011 Mar 17;286(19):16574–16582. doi: 10.1074/jbc.M111.226092

FIGURE 1.

FIGURE 1.

Inhibition of in vitro fusion of GLUT4 vesicles with plasma membranes by N-terminal AS160 constructs and 14-3-3-quenching inhibitors. A, rat N-terminal AS160 PTB domain construct. Shown is the predicted domain organization of rat AS160 transcript variant 4 (LOC686547). The protein sequence was subjected to a domain search using the Pfam tool (42). The diamonds indicates predicted Akt phosphorylation sites. Ser-326 and Ser-350 in the rat sequence correspond to Ser-318 and Ser-341 in the human sequence. B, effect of the human truncated form of AS160 R363X on insulin-stimulated fusion activity. Immunoisolated GLUT4 vesicles from basal cells were pretreated with increasing amounts of FLAG-AS160 R363X for 1 h at 4 °C before measuring the fusion activity in the presence of plasma membranes and cytosol from insulin-stimulated cells. Results are means ± S.E. from four independent experiments. *, p < 0.05 (comparison with the insulin control). C, inhibition of fusion activity by rat N-terminal AS160 constructs. GLUT4 vesicle fusion activity was determined after pretreating immunoisolated GLUT4 vesicles from basal adipocytes with increasing amounts of recombinant His-tagged N-terminal AS160(1–290), AS160(230–532), or AS160(1–532) for 1 h at 4 °C. Fusion activity was determined in the presence of plasma membranes and cytosol from insulin-treated adipocytes. Results are means ± S.E. from four to eight independent experiments. *, p < 0.05 (comparison with the insulin control). D, dose response for the inhibition of fusion activity by His-AS160(1–532). Immunoisolated GLUT4 vesicles isolated from basal cells were pretreated with increasing amounts of His-AS160(1–532) for 1 h at 4 °C before measuring the fusion activity in the presence of plasma membranes and cytosol from insulin-stimulated cells. Results are means ± S.E. from four to five independent experiments. E, effect of 14-3-3-quenching inhibitors R18 and fusicoccin on the in vitro fusion activity. Insulin-stimulated cytosol was pretreated with the indicated concentrations of R18 or fusicoccin for 1 h at 4 °C. Fusion activity was determined in the presence of plasma membranes and cytosol from insulin-treated adipocytes. Result are means ± S.E. from five independent experiments. *, p < 0.05 (comparison with the insulin control).

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