Anti ezh1

Cells were lysed in RIPA lysis buffer (50 mM Tris-HCL [pH 8], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors) for 30 min at 4°C. Protein extracts were resolved by SDS-PAGE and blotted to Nitrocellulose membranes and probed with the following antibodies: anti-beta Actin (Sigma-Aldrich, A5441), anti–EZH2 (AC22, Cell Signaling Technology, 3147S), anti-EZH2 (D2C9, Cell Signaling, 5246), anti-pT487 EZH2 (Abcam, ab109398), anti–SUZ12 (Cell Signaling Technology, 3737S), anti–EED (Millipore, 09-774), anti-EZH1 (Abcam, ab13665), anti-total H3 (Abcam, ab39763), anti-H3K27me3 (Cell Signaling Technologies, 9733), anti-V5 (Abcam, ab9116), anti-total CDK1 (Cell Signaling Technologies, 9112S), anti-pCDK1 (Cell Signaling Technologies, 9111S), anti-MRP1/ABCC1 (Santa Cruz, sc-18835), anti-Ubiquitin (Abcam, ab7780), anti-HOXB7 (Abcam, ab51237), anti-HOXA9 (Abcam, ab140631), anti-TRIM21 antibody (Abcam, ab91432), anti-HSP90 antibody (Abcam, ab13495) and anti-STIP1 antibody (Abcam, ab126724).
Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD).

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).