MCF7 cells were grown in DMEM medium, supplemented with 8% FBS and standard antibiotics. Three days prior to hormonal treatment, cells were switched to phenolred-free DMEM, supplemented with 5% charcoal-treated FBS and standard antibiotics. Medium was replaced daily. Serum-deprived MCF7 cells were treated with 10-7 M Estradiol in 20ml medium for 2 hours and 50 minutes, after which cells were fixed with1% PFA and processed as described (Schmidt et al., Methods 48:3 pp240-2482009). Cell lysis and precipitation procedures were performed as described (Schmidt et al., Methods 48:3 pp240-248 2009).