Ab12073

Cells were lysed for Western blot and immunoprecipitation experiments in the following buffer: 150 mM NaCl, 20 mM Tris (pH 7.4), 5 mM EDTA, 1% Triton, protease arrest (EMD) and phosphatase inhibitors (Calbiochem). To perform immunoprecipitations in the presence of benzonase, cells were lysed in the BC-300 buffer: 20 mM Tris (pH 7.4), 10% glycerol, 300 mM KCl, 0.1% NP-40. The cleared lysate was treated with MgCl₂ to 2.5 mM and benzonase was added at 1250 U/mL. The lysate was incubated for 1 hour with rotation and the reaction was terminated by adding 5 mM EDTA. DNA digestion was confirmed by running lysate on an ethidium bromide gel before setting up the immunoprecipitation experiment. Antibodies used included: BAP1 (C-4; Santa Cruz sc-28383; 1:1000), EZH2 (Active Motif, 39933, Active Motif, 39901, or Millipore, 07-689;1:10,000), SUZ12 (Abcam, Ab12073, 1:1000), ASXL1 (N-13; Santa Cruz sc-85283, 1:1000), L3MBTL2 (Active Motif, 39569, 1:1000), Myc-Tag (Cell Signaling, 2276; 1:2000), SETD8 (ab3798, 1:1000), Tubulin (Sigma, T9026, 1:10,000), H3K27me3 (Abcam, 6002 or Millipore, 07-449, 1:1000), H3 (Abcam, Ab1791, 1:10,000), and H4K20me1 (Abcam, Ab9051, 1:1000).