Figure 1

BRCA1 expression and GABP alpha/beta activity is reduced in SK-BR-3 cells. (a) Western blot analysis of BRCA1 protein levels in MCF-7 and SK-BR-3 cells. Equal quantities of proteins were loaded and levels of TATA Binding Protein (TBP) are shown as an internal control. (b) Quantitative RT-PCR for BRCA1 was carried out for a variety of breast cell lines using TBP as an internal control. Levels are shown relative to MCF-7 cells with the mean and standard deviation of three replicates shown. (c) The relative activities of the BRCA1 proximal promoter construct (L6-pRL) in MCF-7 and SK-BR-3 cells were compared using normalization with an internal control plasmid. For all transfection experiments reported here, the mean and standard deviation of 3 replicates are indicated. Independent experiments were performed a minimum of three times. (d) A ChIP assay was performed using MCF-7 and SK-BR-3 chromatin and antibodies against acetylated histone H3K9 (acH3), haemagglutinin (HA, negative control), RNA polymerase II (RNA pol II) and histone deacetylase I (HDAC). PCR products obtained using primers specific to the BRCA1 promoter (refer to Methods) are shown. (e) The BRCA1 L6-pRL construct was co-transfected with a small hairpin RNA expression construct directed against GABP alpha (shGABP alpha), or the empty H1-2 vector (Empty vector). Results are expressed in relation to the vector transfected cells, for each cell line. (f) Expression vectors for both GABP alpha and beta (GABP alpha/beta) were cotransfected with the L6-pRL promoter construct in both cell lines. Results are expressed in relation to the empty vector controls for each cell line.