H3 binding and methylation by ASCOM. (A) HiTrap Q column fractions containing ASCOM (fractions 40 to 44 in Fig. 2A) were incubated with either agarose beads coupled to histones or oligo(A) (Sigma), separated by SDS-7% PAGE, and probed with anti-ASC-2 (αASC-2) antibody (W). s and p indicate supernatant and precipitate, respectively. Ten percent of the total reaction mixture was loaded as input. (B) The GST pull-down experiments were done as previously described (17, 19) with GST alone or GST fusions to H2A, H2B, H3, and H4, and 20% of the total reaction mixture was loaded as input. (C) HiTrap Q column fractions containing ASCOM (fractions 40 to 44) were immunoprecipitated with the indicated antibodies and measured for methylation activities with bovine serum albumin, H3, or H4. αHA, anti-HA; αALR, anti-ALR; αASH2, anti-ASH2.