Histone Western blotting was performed essentially as above. Protein samples were extracted from ES cells grown on gelatinized plates in ES cell growth media containing 100 U/ml LIF using TriReagent according to manufacturer's procedures (Sigma). 50 µg of total protein was resolved by 15% SDS PAGE and transferred to PVDF (Biorad) using 12.5 mM Tris, 96 mM glycine pH 8.3, 20% methanol transfer solution for 1 hour at 200 mA and 25 V limits. Blots were probed with rabbit polyclonal antibodies to H4 ac-K5, H4 ac-K8, H4 ac-K12 (Serotech), H4 3me-K20, H4 acK16, H3 3me-K9, H3 3me-K27 (Upstate), g-H2AX, H3 3me-K4, H3 ac-K14, H3 ac-K18 (abcam) at 1∶2000 dilutions overnight at 4°C then anti rabbit HRP (Amersham) at 1∶4000 dilution for 1 hrs at RT in PBS with 5% NFDM and 0.1% tween 20 through out. Detection was preformed using Pico signal ECL according to manufacturer's procedures (Pierce).
