Anti eed

DMSO or inhibitor treated cells were washed twice with 1X PBS and whole cell lysates were prepared using lysis buffer (20 mM HEPES, pH 7.8, 150 mM NaCl, 10 mM EDTA, 2 mM EGTA, and 2 mM dithiothreitol, and 0.1% Nonidet P–40). The cell lysates were mixed with Dynabeads conjugated with anti–EZH2 antibody (Cell Signaling) and rotated at 4 °C overnight before removal of the supernatant. The resulting samples were analyzed by Western blot analysis using anti–SUZ12 (Cell Signaling) and anti–EED (Milipore) antibodies.

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).