Sub-populations of c-Myb in MCF-7 cells

We compared two independent c-Myb antibodies during cellular confluence and estrogen stimulation to determine endogenous c-Myb target genes. Interestingly, we discovered that c-Myb specificity was estrogen dependent and multiple antibodies recognize distinct sets of endogenous targets suggesting that epitope masking and domains within the c-Myb protein play a critical role in the activity of the c-Myb protein. MCF-7 cells were plated to 90% confluence in DMEM supplemented with 10% FBS for 24 hours. Cells were subjected to ChIP assay with anti-c-Myb (Upstate (Millipore)) or VKN immune serum (independently developed) and analyzed for enrichment at known c-Myb targets. These samples were compared to ChIP assays performed on cells plated to a high density in DMEM supplemented with 5% Charcoal stripped serum for 48 hours and subsequently induced for 24 hours with purified 17-beta-estradiol. Verified ChIP samples were linearly amplified with a modified version of the Affymetrix whole genome amplification protocol and amplified DNA (6ug) was hybridized to human promoter tiling array 1.0R. Analysis was performed with MAT software for promoter tiling array analysis.