Western blot analysis Whole cell lysates were collected by adding SDS sample buffer. After extensive sonication, the samples were boiled for 10 min and subjected to SDS PAGE. The proteins were then transferred to nitrocellulose membranes and analysed truly by immunoblotting. Immunoprecipitation and MALDI TOF mass spectroscopy For immunoprecipitation assays, approximately 2. 5 107 MDA MB 231 cells were lysed in 500 ul of lysis buf fer supplemented with protease and phos phatase inhibitor cocktails, 2. 5 uM trichostatin, and 50 uM 2PCPA. The cell extracts were cleared by centrifuga tion and then diluted with 500 ul of dilution buffer supplemented with protease and phosphatase inhibitor cocktails, DNase I, 2. 5 uM trichostatin and 50 uM 2PCPA.

The extracts were pre cleared by 30 min incubations with 20 ul of PureProteome Protein G Magnetic Beads at 4 C with rotation. The E2F1 antibody was then cova lently coupled to Dynabeads and added to the pre cleared extracts. After immunoprecipita tion and elution, the bound proteins were digested with trypsin according to standard procedures. The data were recorded and processed with Agilent MassHunter Workstation Software to obtain the Peptide Mass Finger print. The resulting PMF mass spectra were searched against the E2F1 protein sequence with car bamidomethylation of cysteine as a fixed modification and phosphorylation of serine residues as variable modifi cation. The peptide mass tolerance was set to 50 ppm, and a maximum of three missed cleavages was allowed. Microscopy Confocal microscopy was carried out using a Leica TCS 4D confocal microscope.

For indir ect immunofluorescence studies, preparations of the cells on glass slides were fixed with cold acetone for 5 min and then washed with PBS. The cells were incu bated with 3% bovine serum albumin for 20 min and then 2 h at room temperature with specific primary antibodies. The cells were washed three times in PBS and incubated for 1 h at room temperature with Alexa Fluor Dyes as sec ondary antibodies. After 3 washes with PBS, the cells were incubated with 0. 01% 4 6 diamidino 2 phenylidene in water for 5 min. To ensure antibody specificity, primary antibodies were replaced with specific IgGs in negative control reactions. Statistical analysis In all experiments, the mean standard deviation values from three to five determinations in triplicate were calculated. Statistically significant differences were evaluated using a Students t test. Differences were con sidered to be statistically AV-951 significant at P 0. 05. Results and discussion 4OHT up regulates ER expression in ER negative breast cancer cells Breast cancer cells are classified as either ER positive or ER negative, depending on the presence or absence of ER, particularly ER.