Figure 3
Identification of bcl-2 as a putative target gene of BP1. (a) Electrophoretic mobility shift assays were performed to detect potential binding of in vitro transcribed and translated Beta Protein 1 (BP1) to a consensus binding sequence located in bcl-2. Binding of BP1 to a 32P end-labeled dsDNA probe containing the putative BP1 binding site and surrounding sequence is observed as a shifted band (arrow). 500× and 1,000× molar excess unlabeled probes or a negative control (NC) sequence lacking a BP1 binding site were added as a cold competitor for BP1 binding. Addition of 1 or 2 μl BP1 antibody (Ab) resulted in a supershift of the original band (arrowhead). Wheat germ extract alone (WG) served as a control. (b) Western blot analysis of Bcl-2 protein expression in MCF7/EV and MCF7/BP1 cell lines. (c) bcl-2 mRNA from each cell line was analyzed by real-time PCR. MCF7/EV and MCF7/BP1 cells were cultured in the presence or absence of TNFα for 72 hours. Data shown represent bcl-2 levels normalized to 18S. *P < 0.0001. (d) Western blot analysis after culture of cell lines in the presence or absence of TNFα for 72 hours.