Figure 2: Nkx2-5 binds to and activates Tdgf1 expression. (A) Identification of three potential NKEs in the 10kb upstream region of the Tdgf1 gene. (B) Sequence of oligonucleotides used for the EMSA is shown where wild type (WT) and the corresponding mutated (Mut) NKE are in red. Competition studies included increasing amounts of unlabeled WT probe (containing the NKE) or the same unlabeled probe with the NKE mutated (Mut). 32P-labeled WT (NKE) oligonucleotide probe was used for the EMSA and revealed a stable Nkx2-5-DNA (NKE) (lane 2) that could be competed with WT cold competitor (lanes 3-4) but not with Mut cold competitor (lanes 5-6). The complex could be supershifted with anti-myc serum (lane 7). (C) ChIP assay for binding of Nkx2-5 to the Tdgf1 promoter. Anti-myc but not the control antibody is capable of IP of the Tdgf1 promoter from C2C12 cells expressing myc-tagged Nkx2-5. (D) A 1.3 Kb Tdgf1 promoter harboring wild type (WT) or mutated (Mut) NKEs was fused to luciferase (luc) reporter gene and was transfected into C2C12 myoblast cells with or without increased amounts of the Nkx2-5 expressing plasmid. Fold change represents luciferase activity normalized to renilla expression; *, p<0.05, n=3.