FIGURE 2.

Metformin stimulates phosphorylation of Src in an AMPK-dependent manner. A, 3T3-L1 preadipocytes were stimulated for the indicated doses with metformin for 6 h. Cell lysates (25 μg) were analyzed via Western blotting (IB) with anti-phospho-Src antibody. Blotting with anti-Src antibody was used as a protein-loading control. Data in the bar graphs represent the mean ± S.E. (error bars) values of the ratios of densities (p-Src/Src) for at least three individual Western blotting experiments. *, p < 0.05 versus basal values. B, 3T3-L1 preadipocytes were stimulated for 30 min with 1 mm metformin in the presence of Src inhibitor PP2 (1 μm). Cell lysates (25 μg) were analyzed via Western blotting with an anti-phospho-Cbl antibody. Blotting with anti-Cbl antibodies was used as a protein-loading control. Data in the bar graphs represent the mean ± S.E. values of the ratios of densities (p-Cbl/Cbl) for at least three individual Western blot experiments. *, p < 0.05 versus basal values. C, 3T3-L1 preadipocytes were stimulated for 30 min with 1 mm metformin in the presence of compound C. Cell lysates (25 μg) were analyzed via Western blotting using an anti-phospho-Src antibody. Blotting with anti-Src antibody was used as a protein-loading control. Data in the bar graphs represent the mean ± S.E. values of the ratios of densities (p-Src/Src) for at least three individual Western blotting experiments. *, p < 0.05 versus basal values. D, 3T3-L1 preadipocytes were immunoprecipitated with the anti-Src antibody, followed by Western blotting using the anti-AMPKα2 or Src antibody. TCL, total cell lysates.