Enhanced chemiluminescence detection

For preparation of soluble protein in cell lysates, cells were first washed twice with ice-cold PBS and sonicated in 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% (w/v), Nonidet P-40 with protease(s) and 25 mM NaF, 2 mM Na₃VO₄, 0.1 mM PMSF, and 20 μg/mL aprotinin. The soluble and insoluble fractions were prepared by centrifugation at 15,000 × g for 15 min at 4°C. The soluble proteins were separated by electrophoresis through SDS-polyacrylamide gels (6, 8, and 15% w/v acrylamide) and then electrophoretically transferred to a PVDF membrane. [Subsequently, each membrane was blocked with 5% (w/v) skim milk in Tris-buffered saline containing 0.1% (w/v), Tween-20 for 1 h at room temperature and then hybridized with the appropriate primary antibody (diluted in tris-buffered saline) with gentle agitation overnight at 4°C. After washing three times with this buffer, each membrane was incubated with an appropriate secondary antibody for 1 h at room temperature. Each immunopositive band was visualized with enhanced chemiluminescence detection (GE Healthcare). The following antibodies and chemicals were used: anti-EZH2 (1:1000; BD), anti-H3K27me3 (1:1000; Abcam), anti-histone H3 (1:1000; Santa Cruz Biotechnology), and anti-α-tubulin (1:5000; Sigma); MG-132 was from Millipore, and 3-deazaneplanocin A-HCl was from Cayman Chemical Company. Images were quantified with Image J software.

The proteins were isolated from the cells using a radioimmunoprecipitation assay (RIPA) buffer along with a protease inhibitor cocktail, 2 mM phenylmethylsulphonyl fluoride (PMSF), and 1 mM sodium orthovanadate. The cell lysates were centrifuged at 16,000× g for 20 min at 4 °C and the protein was quantified. Equal amounts of proteins were resolved by SDS-PAGE using 6–15% resolving gels and electro-transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% normal horse serum (NHS) in TBS-T and then incubated overnight at 4 °C with primary antibodies specific for NR2B (1:200; Santa Cruz Biotechnology), p-CREB (1:500; Santa Cruz Biotechnology), CREB (1:500; Santa Cruz Biotechnology), cleaved caspase-3 (1:150; Merck Millipore, CA, USA), p53 (1:500; Santa Cruz Biotechnology), and GAPDH (1:1000; Santa Cruz Biotechnology) as well as horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat (Santa Cruz Biotechnology). The protein bands were visualized by enhanced chemiluminescence detection (GE Healthcare, Buckinghamshire, UK) and quantified using ImageJ software 1.53t, Bethesda, MD, USA). All the bands were normalized with GAPDH and p-CREB was normalized with CREB.

Cell lysates were prepared in RIPA buffer in the presence of 1% proteinase inhibitor. Proteins (40 μg) were separated on 8%-15% SDS-PAGE and transferred to PVDF membranes. After blocking using 5 % BSA, membranes were incubated with primary monoclonal antibodies: ABCB1 (1:1000) and β-actin (1:2500) overnight at 4 °C and then incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h. Bands were detected using enhanced chemiluminescence detection (GE Healthcare Lifesciences, Pittsburgh, PA, USA).