Figure 2. Transcription factors SRF and TFAP2 bind to the FXN promoter region in vitro.

EMSA analysis was performed to investigate binding of TFAP2 (A) and SRF (B) to the promoter region of FXN in vitro. Nuclear extracts from HEK293 cells were incubated with [γ-³²P]-ATP labeled oligonucleotides coding the predicted TFAP2 or SRF binding site on the FXN promoter region of interest for 1 hour at 4°C. The binding products were resolved in native polyacrylamide gels (see MATERIALS AND METHODS). Specific competitor (non-radioactive oligonucleotide, so called cold probe, 10 µM) or non-specific competitor (poly(dI-dC), 0.5 mU/µL) was added to assess the specificity of the binding of SRF or TFAP2. Antibodies of TFAP2 (2 mg/ml) and SRF (0.5 mg/ml) were added for supershift, respectively. I: SRF antibody purchased from Santa Cruz Biotech.; II: SRF antibody purchased from Active Motif. A: HEK293 cell nuclear extracts, prepared by the authors, B: Jurkat cell nuclear extracts, purchased from Active Motif (Carlsbad, CA).