Immunoprecipitation and Western Blot Analysis

The assay was performed as described previously (18 (link)) with minor modifications. Briefly, 600 μl nuclear extracts (NE) or whole cell extracts (WCE) were pre-cleared by incubation with 50 μl protein A-beads for 1 h at 4°C and the supernatants were incubated with 50 μl anti-HA or anti-Flag agarose beads (Sigma) for 2 h. The beads were washed twice with buffer D (20 mM HEPES–KOH [Ph7.9], 15% glycerol, 0.2 mM EDTA, 0.1% NP-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) containing 0.3 M KCl and then twice with buffer D containing 0.1 M KCl. The precipitates were eluted off the beads by incubation with 0.1 M glycine (pH 2.5) and analyzed by Western blotting with the indicated antibodies.