FIGURE 6.

IRAP can be cross-linked to Glut4 in 3T3-L1 cells. A, differentiated G adipocytes stably expressing myc₇-Glut4 were incubated with DSP, and cell lysate (1.5 mg) was immunoprecipitated (IP) the anti-Myc antibody or nonspecific IgG and protein G. Proteins were eluted with Laemmli sample buffer with 50 mm dithiothreitol at 37 °C for 30 min. B, wild type 3T3-L1 adipocytes were incubated with DSP, and cell lysate (1.5 mg) was immunoprecipitated with the anti-Glut4 monoclonal antibody 1F8 or nonspecific IgG. C, wild type 3T3-L1 adipocytes were incubated with DSP or DMSO as described under “Experimental Procedures,” and cell lysate (50 μg) was analyzed by PAGE (without reducing agents) and Western blotting with the polyclonal antibody against cellugyrin. D, S and GS pre-adipocytes were incubated with DSP, and cell lysates (0.15 mg each) were immunoprecipitated with the anti-Myc antibody or nonspecific IgG and protein G. Input lanes show Western blot analysis of 15 μg of total lysates.