Cebpd sc 636

The experimental cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM DTT, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, aprotinin 1 μg/ml, leupeptin 1 μg/ml and 1 mM Na₃VO₄). The proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies as follows: AR (pg-21, Millipore, Billerica, MA, USA), CEBPD (SC-636, Santa Cruz, Dallas, TX, USA), DNMT1 (IMG-261A, IMGENEX, San Diego, CA, USA), DNMT3A (IMG-268A, IMGENEX), DNMT3B (IMG-184A, IMGENEX), E2F1 (SC-193, Santa Cruz), EZH2 (07-400, Upstate, Billerica, MA, USA), HA (MMS-101R, COVACE, Princeton, NJ, USA), H3K27 trimethylation, (07-449, Upstate), myc (SC-40, Santa Cruz), CASP8 (RB-1200, Thermo Scientific, Pittsburgh, PA, USA), CASP3 (9661, Cell Signaling Technology, Danvers, MA, USA), SUZ12 (07-379, Upstate), tBid (2002, Cell Signaling Technology) and α-tubulin (T6199, Sigma). Mitochondrial protein and cytosolic protein were isolated using the Cytochrome c Releasing Apoptosis Assay kit according to the manufacturer's instructions (K257-100, BioVison, San Francisco, CA, USA).