H2AK119u1 pull downs

WT and uH2A dinucleosomes were incubated with prewashed, magnetic, streptavidin coated beads (Fisher) for 1 h at 4 °C, and then washed five times with 250 mM NaCl, 10 mM Tris-HCl pH 7.0 and 0.01% Tween 20. 10 μg dinucleosomes (WT or uH2A) were incubated with 250 ug nuclear cell extract for 1 h at 4 °C, again washed five times with 250 mM NaCl, 10 mM Tris-HCl pH 7.0 and 0.01% Tween 20, and finally the reaction stopped by adding SDS loading buffer. Samples were analysed by SDS-PAGE and western blot. Primary antibodies used were anti-HA (Roche 3F10 11867423001) at 1:1,000 dilution, anti-ATRX (a gift from R.Gibbons) at 1:10 dilution, anti-EZH2 (Cell Signalling 5246) at 1:1,000 dilution, anti-JARID2 (Novus Biologicals NB100-2214) at 1:1,000 dilution and anti-H4 (Abcam AB7311).