MCF7 cells (2 × 108) were suspended in nuclear/cytoplasmic separation buffer (20 mM tris-HCl at pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, and 0.1 mM EDTA supplemented with RNase inhibitors and protease inhibitors) and lysed by a dounce homogenizer. After centrifugation at 1000 rpm for 10 min, nuclear pellets were resuspended in a buffer containing 25 mM tris at pH 7.6, 0.2 mM EDTA, 5 mM MgCl2, 0.1 M KCl, 10% glycerol, 0.5 mM DTT, 1 mM benzamidine, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and RNase inhibitors. Nuclear extracts were applied to a Superose 6 gel filtration column and an FPLC system (GE Healthcare) and calibrated with protein standards (blue dextran, 2000 kDa; thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 158 kDa; bovine serum albumin, 75 kDa; all from Amersham Biosciences). The column was eluted at a flow rate of 0.5 ml/min and fractions were collected. Samples were subjected to TRAP, Western blot, and reverse transcription PCR analysis.

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