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. 2007 Jul 23;27(19):6818–6831. doi: 10.1128/MCB.00375-07

FIG. 6.

FIG. 6.

TRB3 inhibits ERK-dependent phosphorylation of C/EBPβ during the early phase of adipogenesis. (A) Effect of TRB3 overexpression in 3T3-L1 cells on insulin-dependent activation of Akt/GSK3 and ERK pathways. Cells were grown to confluence and induced with 100 nM insulin for 5 or 10 min. Protein extracts were prepared at the times indicated. A total of 30 μg of total protein extracts was loaded per lane and analyzed by Western blotting. (B) Effect of TRB3 overexpression in 3T3-L1 cells on early activation of Akt/GSK3 and ERK pathways by the adipogenic cocktail. Cells were induced to differentiate, and protein extracts were evaluated at the times indicated. A total of 30 μg of total protein extracts was loaded per lane and analyzed by Western blotting. The results are representative of three independent experiments. (C) TRB3 regulation of C/EBPβ phosphorylation in nuclear extracts of 3T3-L1 preadipocytes. Nuclear and cytoplasmic extracts were prepared at 24 h postinduction, and 30 μg of protein were loaded in each lane and analyzed by Western blotting. Nuclear and cytoplasmic extract quality was determined with lamin A and SOD4 protein. The results are representative of three independent experiments. Quantification of the phosphorylation of C/EBPβ in 3T3-L1 preadipocytes nuclear extracts. Western blot results were analyzed with numeric quantification program (ImageQuant). The results are representative of the three independent experiments presented in Fig. 5B and are expressed as the ratio of phosphorylated C/EBPβ on total C/EBPβ.