Figure 3.

(A) Syntaxin-4 is present in 3T3-L1 adipocytes and associates with VAMP-2. Detergent extracts of 3T3-L1 adipocytes were subjected to immunoprecipitation (IP) with a nonimmune antiserum (NI) or anti-syntaxin-4 (αSynt4). The immunoprecipitated proteins were separated by SDS/PAGE and immunoblotted (IB) with either αSynt-4 or αVAMP-2. (B) Effect of various GST-fusion proteins on insulin-regulated GLUT4 translocation. Permeabilized adipocytes were incubated in buffer alone or in the presence of 10 μM GST alone, GST–VAMP-2, or GST–syntaxin-4 for 10 min. The cells were then stimulated with insulin for an additional 10 min followed by analysis of GLUT4 translocation by immunofluorescence of plasma membrane sheets.