Ecl western blotting detection regents

Immunoblotting analysis was performed as previously described^{65 (link)}. Cells were washed twice with ice cold PBS, and harvested in protein lysis buffer (50 mM HEPES [pH7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 1× complete protease inhibitor tablet [Roche]). Total cell lysates were separated on 4-12% Bis-Tris gels (Life Technologies), transferred onto PVDF membrane (Merck Millipore). The blots were probed with antibodies for H3, H3K4me2, H3K9me2, H3K27me2 (#9847 from Cell Signaling Technologies for above antibodies; 1:1000), H3K27me3 (Cell Signaling Technologies; #9733, 1:1000), SUZ12 (Cell Signaling Technologies; #3737, 1:1000), Ezh2 (Cell Signaling Technologies; #5246, 1:1000), RpAb46/48 (Santa Cruz Biotechnology; #33170, 1:1000), EED (Santa Cruz Biotechnology; #28701, 1:1000), p-S6K (Thr389, Cell Signaling Technologies; #9234, 1:1000) and S6K (Cell Signaling Technologies, MA, USA; #9202, 1:1000) as indicated in figures. Protein signals were detected using HRP conjugated secondary antibodies and enhanced chemiluminescence (ECL) western blotting detection regents (Thermo Fisher Scientific, MA, USA).