Protein samples were separated by electrophoresis on denaturing 4–12% premade polyacrylamide gels (Invitrogen) and blotted to PVDF membranes (Millipore). Membranes were blocked in TBST buffer plus 5% milk. Major antibodies: H3K27me3 (Cell Signaling, 9733), H3K27me2 (Millipore 07–452) and H3K27me1 (Millipore, 07–448), H3R8me2s (Epigentek A-3706–050), PRMT5 (Millipore 07–405), EZH2 (Cell Signaling 5246), Symmetric Di-methyl Arginine (Cell Signaling 13222), UTX (Cell Signaling 33510), EED (Millipore, 09–774), RBBP4 (Bethyl Laboratories A301–206A-T) and SUZ12 (Cell Signaling 1335947).
Histones were purified from mouse BM cells using Histone Extraction Kit (Active Motif, 40028) as per manufacturer's instructions. Successful purification of histones was confirmed by Coomassie staining before they were used for western blotting.
Histones were purified from mouse BM cells using Histone Extraction Kit (Active Motif, 40028) as per manufacturer's instructions. Successful purification of histones was confirmed by Coomassie staining before they were used for western blotting.