Figure 4

The effect of Oct-1 decoy oligonucleotide treatment on binding of Oct-1 transcription factor to the endogenous γ-globin gene promoter region DNA in K562 cells. The K562 cells were treated with either the decoy or the scrambled oligonucleotides for four days. The cells were fixed in 1% formaldehyde and sonicated to lyse the cells. An immunoprecipitation protocol was performed to pull down the Oct-1 transcription factor using an Oct-1 antibody and the Protein A-conjugated agarose beads. Half of the beads were analyzed by western blots to determine the level of Oct-1 transcription factor precipitated by the IP and the rest of the beads were treated in 5 M NaCl at 65°C for four hours to reverse the protein-DNA crosslinks and the recovered DNA was analyzed by a quantitative PCR (qPCR) protocol to determine the level of γ-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. A pair of primers that bind to the γ-globin gene at the -350 to -330 and +50 to + 30 region sequences respectively was used in the qPCR study. (A) Detection of the Oct-1 transcription factor precipitated by the IP protocol. (B) Quantification of the levels of γ-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor in the IP assay. The results are from three independent experiments.