Mouse anti h3

Total cell lysates were resolved in 4-12% Bis-Tris Protein Gels (Invitrogen) with MOPS buffer (Invitrogen) and transferred in for 1.5h (80 V) onto PVDF membrane (Millipore) in Tris-Glycine transfer buffer containing 20% MeOH and 0.05% SDS. Blots were blocked for 1 h in blocking buffer (5% milk in 0.1% Tween 20 PBS) and incubated with primary antibodies: mouse anti-H4 (1:1000, Abcam #174628), rabbit anti-H4R3me2a (1:1000, Active Motif #39705), mouse anti-FLAG (1:5000, Sigma #F1804), mouse anti-β actin (1:3000, Abcam #3280), mouse anti-H3 (1:1000, Abcam #174628), rabbit anti-H3R2me2a (1:1000, Millipore #04-808), rabbit anti-Rme2a (1:1000, Cell Signaling Technology #13522), rabbit anti-Rme2a Asym24 (1:1000, Millipore#07-414), rabbit anti-Rme2S (1:1000, Cell Signaling Technology #13222), rabbit anti-Rme1 (1:1000, Cell Signaling Technology #8015) in blocking buffer overnight at 4 °C. After five washes with 0.1% Tween 20 PBS the blots were incubated with goat-anti rabbit (IR800 conjugated, LiCor #926-32211) and donkey anti-mouse (IR 680, LiCor #926-68072) antibodies (1:5000) in Odyssey Blocking Buffer (LiCor) for 1 h at RT and washed five times with 0.1% Tween 20 PBS. The signal was read on an Odyssey scanner (LiCor) at 800 nm and 700 nm.