C/EBPα expression suppresses the BPl-TIRE-Luc reporter activity in H4IIE cells, independently of the phosphorylation status of T222/T226. (A) H4IIE cells were transfected with BPl-TIRE-Luc (10 μg) or DM5-Luc (10 μg) along with either pcDNA6 vector (1 μg), Flag-C/EBPα (1 μg) or Flag-C/EBPα-AAA (1 μg). Subsequently, cells were incubated with or without 10 nM insulin for 20 h prior to lysis and luciferase assay. Results are presented as relative luciferase activity to the control (serum free) and are average ± SEM (n = 3) from two independent experiments. (B) H4IIE cells were infected with adenovirus expressing either β-Galactosidase (AD-βgal) or active GSK3β (Ad-GSK3 S9A) for 20 h prior to transfection as described in (A). Results are presented as relative luciferase activity to the control (serum free) and are average ± SEM (n = 6) from two independent experiments. *** p < 0.001 compared to control (ad-b-gal).