Anti ezh2

FACS sorted CML LSK cells were treated as described in the results or directly lysed in buffer containing 0.5% Nonidet P-40 (Sigma Diagnostics) and 0.5% sodium deoxycholate supplemented with phenylmethylsulfonyl fluoride (1 mM/L), protease inhibitor mixture, and phosphatase inhibitors (50 mM/L sodium fluoride, 1 mM/L sodium vanadate; all from Sigma Diagnostics). Proteins were resolved on 4% to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to nitrocellulose membrane. Primary antibodies used were anti–EZH1 (cat: ab176115; Abcam, Cambridge, MA), anti–EZH2 (cat: 39933; Active Motif, Carlsbad, CA), anti– H3K27me3 (cat: ab6002; Abcam, Cambridge, MA), anti-actin (clone: AC15; cat: A5441; Sigma Aldrich, St. Louis, MO), anti-H3 (cat: ab1791; Abcam, Cambridge, MA), anti-P-ERK1/2 (Thr202/Tyr204) (cat: 4370S; Cell Signaling, Danvers, MA), anti-P-AKT (Ser473) (cat: 4060S; Cell Signaling, Danvers, MA), and Horseradish peroxidase– conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibody detection was performed by using the SuperPico and SuperFemto kits (Pierce Biotechnology, Rockford, IL).

Cell pellets were homogenized in RIPA buffer (Pierce, Rockford, IL) with complete protease inhibitor (Roche, Basel, Switzerland). Insoluble material was removed by centrifugation at 16,000 g at 4oC for 15mins. Protein concentrations were determined using the Bradford Protein Assay Kit (Bio-Rad, Hercules,CA). Equal amounts of protein were separated on 4%–12% NuPAGE SDS gel (Invitrogen, Grand Island, NY) and transferred onto a PVDF membrane. anti-H3K27me3, anti-H3K27me2, anti-H4K16ac, anti-H3K27ac, anti-H3K79me1, anti-H3, anti-H4, anti-WT1 and anti-EZH2 (1:1000 dilution, Active Motif), anti-PAX8 (1:1000, Abcam) and anti-B-actin (1:10,000 dilution, Sigma-Aldrich, St. Louis, MO) were used. Bands were visualized using the ECL Kit (Pierce, Rockford, IL).

Nuclear extracts from ROS17/2.8 cells, grown in the presence or absence of 10⁻⁸ M 1,25(OH)₂D₃ or vehicle, were prepared as previously described (Paredes et al., 2004 (link)). Protein levels were quantified using Bio-Rad Protein Assay reagent according to manufacturer’s instructions and using bovine serum albumin as standard (Bio-Rad, CA, USA). For western blot analyses, 10 μg of total protein were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to a nitrocellulose membrane. Immunoblot was performed using the following antibodies: anti-TFIIB C-18 (sc-225, Santa Cruz Biotechnology, TX), anti-EZH2 (39901, Active Motif, CA) and anti-UTX/KDM6A (ab91231, Abcam). Immunoblotting was carried out using secondary antibodies conjugated to horseradish peroxide (Santa Cruz Biotechnology) and substrates for enhanced chemiluminescence (Thermo Scientific, IL, USA).