Anti suz12

Human tissues for Western blotting were lysed in RIPA (50 mM Tris pH 7.4, 1% NP40, 150 mM NaCl, 40 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA and 10 μl/ml of protease inhibitor cocktail) buffer. For cell lines, the cells were seeded in dishes at 60% confluence overnight and then treated with emodin or vehicle control for 24 h. The cells were lysed in ice-cold RIPA buffer according to previously described methods [56 (link)]. Protein concentrations were quantified using a BCA assay (Thermo Scientific Pierce) according to the manufacturer's protocol. Lysate 5-40 μg was loaded into each lane by Western blotting. The specific signal was detected using a secondary antibody coupled with horseradish peroxidase (Jackson ImmunoResearch Labs) and Chemilucent Plus Western Blot Enhancing Kit (Millipore, Bedford, MA, USA).
The antibodies used included anti-EZH2 (Clone: AC-22, CellSignaling, Danvers, MA, USA), anti-Actin (Sigma, St Louis, MI, USA), anti-V5 tag (AbD Serotec, USA), anti-H3K27me3 (Merck-Millipore, Upstate, Lake Placid, NY, USA), anti-EED (Abcam, Cambridge, UK), anti-Suz12 (CellSignaling, Danvers, MA, USA), anti-HA (Clone:12CA5, Roche), and anti-GAPDH (Sigma, St Louis, MI, USA). Secondary antibodies, including goat-anti-mouse-HRP and goat-anti-rabbit-HRP, were purchased from Jackson ImmunoResearch Labs (Westgrove, CA, USA).

Two wells (of a 12-well plate) of confluent CMs were fixed in 1% formaldehyde for 10 min and then quenched with 0.125 M of glycine for 5 min. Chromatin was sonicated with Bioruptor Diagenode Sonicator and immunoprecipitated overnight at 4 °C with 4 μg of Lamin A/C antibody (anti Lamin A/C (636), Santa Cruz sc-7292×) or 5 μg of antibodies against the analysed histone modifications (anti-H3K4me3, Active Motif 39159; anti-H3K27me3, Abcam ab6002; anti-H3K9me3, Abcam ab8898; anti-Suz12, Cell Signaling, #3737).
The pathology tissue chromatin-immunoprecipitation (PAT-ChIP) procedure was carried out as previously described^{69 (link)}. In brief, paraffin was removed with fresh Histolemon solution (Carlo Erba), chromatin extracted, sonicated and immunoprecipitated. For the immunoprecipitation step 3 µg of ChIP-grade antibody was added to 0.5–2.5 µg of tissue chromatin.
Quantitative PCR was performed in triplicate using SYBR Select Master Mix (ThermoFisher Scientific). Ct values were calculated using Viia^TM 7 software and relative enrichment was calculated as ChIP/input ratio. Primer sequences are listed in the Supplementary Table 6.

ESCs were grown on slides without feeders, fixed with 2% formaldehyde for 15 min, permeabilized with 0.4% Triton X-100 for 5 min and blocked with 0.2% fish gelatin (Sigma) for 30 min. Slides were incubated with primary antibody for 2 h (diluted in 0.2% fish gelatin and 5% normal goat serum), washed three times and incubated with Alexa-fluor conjugated secondary antibody for 2 h (Life Technologies). After washing five times, the slides were stained with DAPI (1 μg ml⁻¹), and mounted using mounting media (Dako). Primary antibodies used were protein-A purified anti-AEBP2 (ref. 36 (link)) at 1:10 dilution, anti-H3K27me3 (Active motif 39157) at 1:500 dilution, anti-SUZ12 (Cell Signalling 3737) at 1:500 dilution, anti-EZH2 (Cell Signalling 5246) at 1:500 dilution, anti-EED (a kind gift from A. Otte) at 1:100 dilution, anti-JARID2 (Novus Biologicals NB100-2214) at 1:500 dilution, H2AK119u1 (NEB 8240) at 1:500 dilution, anti-FLAG (Sigma M2 F1804 and F7425) at 1:500 dilution, anti-mCherry (Source Bioscience ABE3523 and gift from F. Barr) at 1:500 and 1:800 dilution, anti-RING1B at 1:500 dilution and anti-ATRX (a gift from R. Gibbons) at 1:10 dilution.

The following antibodies were used for Chromatin Immunoprecipitation (ChIP), RNA-binding protein Immunoprecipitation (RIP) or immunoblotting: anti-H3K27me3 (17–622, Millipore), anti-H3K9me3 (ab8898, Abcam), anti-EZH2 (07–689, Millipore), anti-SUZ12 (3737, Cell Signaling Technology), anti-EED (17–663, Millipore), anti-GAPDH (M20006, Abmart).

The cells were lysed using Western and IP lysis buffer (Beyotime Biotechnology) and the cell lysate was incubated with 40 μL of protein-A/G agarose beads (Millipore), 5 μg anti-EZH2, anti-SUZ12 (Cell Signaling Technology, CST) or anti-DDX5 (proteintech#67025-1-Ig) antibodies at 4 °C overnight. After washing five times with phosphate buffered saline (PBS) buffer, the samples were analyzed by Western blotting or MS.