Full-length cDNA of Br140 was cloned into vector pBacPAK8 or pBacPAK8 carrying an N-terminal His-Flag tag. Full-length cDNA of elg1 was cloned into vector pBacPAK8 carrying an N-terminal double HA tag. Purification of the recombinant Enok and Br140 from Sf21 cells was performed as described previously (Huang et al. 2014 (link)).
Short oligonucleosomes were purified from HeLa cells as described previously (Utley et al. 1997 (link)). Reconstitution of recombinant nucleosomes and in vitro HAT assay were carried out essentially as described in the previous study (Huang et al. 2014 (link)). Briefly, nucleosomes were incubated with or without recombinant Enok and/or Br140 in HAT buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 100 µM EDTA, 10 mM sodium butyrate, 5% glycerol, 1 mM DTT, 100 µg/mL BSA, 1 mM acetyl-CoA, 1 mM PMSF) for 2 h at 27°C. For autoradiography (Fig. 1 C, bottom two panels), 0.4 µCi of 3H-acetyl-CoA (PerkinElmer) instead of 1 mM acetyl-CoA was used in 20-µL reactions, with a final concentration of 100 µM. The reactions were then adjusted using 2× SDS sample buffer to a final concentration of 1× (62.5 mM Tris-HCl at pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue, 143 mM β-mercaptoethanol) and heated for 6 min at 98°C followed by Western blotting or autoradiography.
Short oligonucleosomes were purified from HeLa cells as described previously (Utley et al. 1997 (link)). Reconstitution of recombinant nucleosomes and in vitro HAT assay were carried out essentially as described in the previous study (Huang et al. 2014 (link)). Briefly, nucleosomes were incubated with or without recombinant Enok and/or Br140 in HAT buffer (50 mM Tris-HCl at pH 8.0, 50 mM KCl, 100 µM EDTA, 10 mM sodium butyrate, 5% glycerol, 1 mM DTT, 100 µg/mL BSA, 1 mM acetyl-CoA, 1 mM PMSF) for 2 h at 27°C. For autoradiography (
