Ring1b

ChIP assays were carried out on ∼2–5 million cells per sample and per epitope, following the procedures described previously (Mikkelsen et al, 2007 (link)). In brief, chromatin from formaldehyde-fixed cells was fragmented to a size range of 200–700 bases with a Branson 250 sonifier. Solubilized chromatin was immunoprecipitated with the indicated antibodies overnight at 4°C. Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After cross-link reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). ChIP DNA was quantified with Qubit. 1–5 ng ChIP DNA samples were used to prepare sequencing libraries, and ChIP DNA and input controls were sequenced with the Nextseq 500 Illumina genome analyzer.
Antibodies used for these studies were SMARCA2/4 (39805; Active Motif), H3K4me1 (ab8895; Abcam), H3K4me3 (07-473; Millipore), H3K9ac (ab4441; Abcam), H3K27ac (39133; Active Motif), H3K27me3 (07-449; Millipore), H3K36me3 (ab9050; Abcam), V5 (ab15828; Abcam), RING1B (5694; Cell Signaling), H2AK119ub (8240; Cell Signaling), RYBP (59451204; Sigma-Aldrich), and USP7 (A300-033A; Bethyl Laboratories).

Cells were washed in ice-cold PBS and lysed in modified RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP‐40, pH 8.0, 1× protease inhibitor cocktail, and phosphatase inhibitors). Lysates were clarified by centrifugation (16,000 × g, 30 min) and subjected to western blotting using the indicated primary antibodies at 1:1000 dilution. Protein-antibody conjugates were visualized using a chemiluminescent detection kit (ThermoScientific). Antibodies against BMI-1, SUZ12, EZH2, Ring1A, Ring1B, and DNMT1 were purchased from Cell Signaling Technologies (Danvers, MA, USA).

Primary antibodies were against p19 (NB200‐106, Novus Biologicals, CO, USA), p21 (#2947, Cell Signaling Technology, Beverly, MA, USA), p53 (#2424, Cell Signaling Technology, USA), p16 (ab211542, Abcam, Cambridge, MA, USA), renin (#5250, Cell Signaling Technology, USA), Ang II (NBP1‐31127, Novus Biologicals, USA), ANP (#AB5490, Millipore, MA, USA; sc‐515701, Santa Cruz Biotechnology Inc., Dallas, TX, USA, USA), BNP (#DF6902, Affinity Biosciences, OH, USA; ab19645, Abcam, USA), GATA4 (#19530, Proteintech, IL, USA; sc‐25310, Santa Cruz Biotechnology Inc., USA), LC3B (#NB600‐1384, Novus Biologicals, USA), p62 (#39749, Cell Signaling Technology, USA), Bmi‐1 (#5856, Cell Signaling Technology, USA; 66161‐1‐Ig, Proteintech, USA), RING1B (#5694, Cell Signaling Technology), NF‐κB‐p65 (sc‐8008, Santa Cruz Biotechnology Inc., USA; #8242, Cell Signaling Technology, USA), p‐p65 (Ser536) (ab76302, Abcam, USA), IκB‐α (AF1282, Beyotime Biotechnology, Shanghai, China), p‐IκB‐α (Ser32) (sc‐8404, Santa Cruz Biotechnology Inc., USA), p‐Chk2 (Thr68) (PA5‐104715, Invitrogen Inc. CA, USA), SOD‐2 (NB100‐1992, Novus Biologicals, USA), HSC70 (10654‐1‐AP, Proteintech, USA), p‐ULK1 (Ser757) (#14202, Cell Signaling Technology, USA) and ULK1 (sc‐390904, Santa Cruz Biotechnology, USA). β‐actin (AP0060, Bioworld Technology Inc., MN, USA) was loading control for total protein.

Cells were washed with ice-cold PBS. Cell pellets were resuspended in RIPA lysis buffer (50mM Tris pH7.5, 150mM NaCl, 150mM NP40, 0.5% Na-deoxycholate, 0.1% SDS, 10% glycerol). Cell lysis was performed for 20 mins on ice. Protein lysate was recovered by centrifugation and protein-containing supernatant was kept. Protein concentration was measured by Bradford assay (BIORAD). Western blotting was performed on 40 μg of total 46C mES cell protein extracts with antibodies raised against EZH2 (Diagenode), RING1B (Cell Signaling), OCT4 (Abcam), NANOG (Abcam) and γ-tubulin (Sigma), all at 1:1000 dilutions. Western blotting was performed with ECL kit (PerkinElmer).

ASCL1 (#43666), BAP1 (#13271S), H3K27ac (#8173S), H3K4me1 (#5326S), H3K4me3 (#9751), H3K27me3 (#9733), H2AK119Ub (#8240), ASXL2 (#71257), and RING1B (#5694) were purchased from Cell Signaling Technology (CST) company. HSP90 (sc-7947) was purchased from Santa Cruz. Tubulin antibody (E7) was purchased from Developmental Studies Hybridoma Bank. FOXK1 (A301-727A), FOXK2 (A301-729A), and HCFC1 (A301-399A) antibodies were purchased from Bethyl-Laboratories. ASXL1 and ASXL3 antibodies were generated as previously described [11 (link)]. EZH2 inhibitor GSK126 was purchased from Cayman (15415).