Fig. 3.

Impact of C/EBPβ expression on TRB3 gene expression A, Ectopic expression of C/EBPβ augments TRB3 expression. Fao cells were transduced with either GFP- or C/EBPβ-expressing pMIGR1 retrovirus, and 48 h later, the cells were treated with insulin at indicated time points and total cell lysates were prepared for Western blot with anti-TRB3 (top), anti-C/EBPβ (middle), and anti-β-tubulin (bottom) antibodies, respectively. B, Transient transfection and luciferase assay to show that C/EBPβ activates TRB3 promoters in HepG2 cells. The experiments were carried out in triplicate and repeated twice. The final luciferase activity was normalized to β-galactosidase activity cotransfected with reporter and effector plasmids. The error bars represent sd from three experiments. *, P < 0.003, Student’s t test; **, P < 0.011. C, Knockdown of C/EBPβ inhibits TRB3 expression induced by insulin Fao cells expressing either control (scrambled) or C/EBPβ shRNA via adenoviral transfer were treated with insulin at indicated times, and total cell lysates were prepared for Western blot with anti-TRB3 (top), anti-C/EBPβ (middle), and anti-β-tubulin (bottom) antibodies, respectively. D, ChIP assay to show that C/EBPβ binds TRB3 promoter Fao cells expressing control (scrambled) or C/EBPβ shRNA were treated with insulin, and ChIP assays were carried out as described in Materials and Methods. E, Transient transfection and luciferase assay to show that C/EBPβ shRNA suppresses TRB3 promoters in HepG2 cells. The experiments were carried out in triplicate and repeated twice. The final luciferase activity was normalized to β-galactosidase activity cotransfected with reporter and effector plasmids. The error bars represent sd from three experiments. *, P < 0.012; **, P < 0.021.