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. 2012 Nov 7;287(53):44121–44129. doi: 10.1074/jbc.M112.361386

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© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 1. — AICAR and metformin induce phosphorylation of c-Cbl in an AMPK-dependent manner. A, 3T3-L1 preadipocytes were stimulated for the indicated times with 1 mm AICAR. Cell lysates (25 μg) were analyzed via Western blotting (IB) using an anti-phospho-Cbl (Tyr⁷³¹) antibody. Blotting with anti-Cbl antibody was used as a protein-loading control. B, 3T3-L1 preadipocytes were stimulated for the indicated doses of metformin for 1 h. Cell lysates (25 μg) were analyzed via Western blotting using an anti-phospho-Cbl (Tyr⁷³¹) antibody. Blotting with anti-Cbl antibody was used as a protein-loading control. C, 3T3-L1 preadipocytes were stimulated for 30 min with 1 mm metformin in the presence of compound C (2 μm). Cell lysates (25 μg) were analyzed via Western blotting with an anti-phospho-Cbl (Tyr⁷³¹) antibody. Blotting with the anti-Cbl antibody was used as a protein-loading control. D, 3T3-L1 preadipocytes were stimulated with 500 μm metformin for the indicated times. Cell lysates (25 μg) were analyzed via Western blotting with an anti-phospho-Cbl (Tyr⁷³¹) antibody. Blotting with the anti-Cbl antibody was used as a protein-loading control. Data in the bar graphs (A–D) represent the mean ± S.E. (error bars) values of the ratios of densities (p-Cbl/Cbl) for at least three individual Western blotting experiments. *, p < 0.05 versus basal values.