Binding of transcription factor NF-κB to human
promoter. (A) Binding of NF-κB transcription factor to Mcl-1-κB probe in vitro. Nuclear extracts of TE-1 cells were prepared for EMSA. Nuclear extracts were preincubated with biotin-labeled Mcl-1-κB oligonucleotide probe in the absence or presence of antibodies directed against different NF-κB subunits p50, p52, p65, c-Rel, RelB or control antibody (IgG) (indicated above each lane) and then gel shift assays were performed. The arrows indicated the DNA-protein complexes. Free labeled probes are also indicated. The results are representative of two independent experiments. NS, nonspecific band. (B) Transcription factor NF-κB p50 and p65 subunits directly interacted with human Mcl-1 promoter in intact cells. The cross-linked chromatin was precipitated with antibody against transcription factor NF-κB subunit p50, p52, p65, c-Rel or RelB. The positive control is represented by the input fraction. Negative controls included a no chromatin sample and a nonspecific antibody (α-IgG) sample. Precipitated DNA was analyzed by PCR using primers that amplified a 200-bp region, which included the NF-κB binding site of human Mcl-1 promoter. Data represented the mean ± S.D. of two separate experiments. Statistical significance: * p < 0.05, compared with the IgG control.