Figure 6. FoxO4 activates Arg1 transcription.

(A) Arg1 promoter (4.8kb)-driven luciferase (Luc) reporter construct (left panel) or 3xIRS-luc (right panel) were co- transfected into 293A cells with the indicated FoxO expression plasmids and internal control CMV-LacZ. The luciferase activities were normalized against co-transfected β-galactosidase and expressed relative to that from vector Flag-pcDNA transfected cells (n=3). (B) One potential Sp1- (open circle) and three FoxO4-binding sites (solid circles) were identified in the Arg1 promoter. Arg1-luc reporters with different 5’ends and point mutations in the Sp1- (m2) and FoxO4-binding site (m1) were used to identify the functional Sp1 and FoxO4 binding sites in the Arg1 promoter (n=3). (C) A gel shift assay was performed with ³²P-labeled oligonucleotide probe containing the Sp1 and FoxO4-binding sequence in the Arg1 promoter and lysates of cells transfected with Flag-FoxO proteins as indicated.