Figure 4
bcl-2 is a direct transcriptional target of BP1. (a) Schematic diagram of bcl-2 P1 promoter constructs. LB170 contains the Beta Protein 1 (BP1) binding site (underlined). delLB170 contains a deletion of seven of the nine nucleotides of the binding site (indicated by X), and mutLB170 contains the mutated BP1 binding site (lowercase, underlined). LUC, luciferase. (b) MCF7/EV and MCF7/BP1 cells were transiently transfected with LB170, delLB170, or mutLB170, as well as a plasmid encoding β-galactosidase. Forty-eight hours post transfection, protein was extracted and assayed for luciferase activity. Relative light units were normalized to β-galactosidase expression units to signify levels of bcl-2 P1 promoter activity (RLU/Bgal). *P < 0.0001, • P < 0.05. (c) Electrophoretic mobility shift assay: in vitro transcribed and translated BP1 protein (BP1) was incubated with a 32P end-labeled DNA oligonucleotide probe containing the sequence from the bcl-2 promoter including the BP1 binding site (arrow). Cold competitor DNAs including a bcl-2 sequence identical to the probe (bcl-2), mutated bcl-2 (mbcl-2), and a negative control (NC) lacking the BP1 binding site, were added at 500× or 1,000× molar excess. Wheat germ extract (WG) incubated with the bcl-2 probe served as a control.