Calf thymus bulk histones

Calf thymus bulk histones (Sigma) were incubated with FLAG-USP11 or FLAG-HDAC2 IPs at 37°C for 90 min in deubiquitination assay buffer (50 mM NaCl, 1mM DTT and 50 mM Tris–HCl, pH 7.5) or histone deacetylation assay buffer (150 mM NaCl, 1mM DTT and 10 mM Tris–HCl, pH 8.0). Each reaction was stopped by mixing with SDS loading buffer followed by western blot analysis with antibodies against H2AK119ub, H2BK120ub, or H3ac.

For deacetylation assay, 10 μg calf thymus bulk histones (Sigma) were incubated with 0.25–5 μg of GST-CS or GST-HDAC3 in deacetylation assay buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM MgCl₂) at a final volume of 15 μl for 2 h at 37 °C. The reaction mixture was boiled in SDS buffer, resolved on SDS-PAGE, and subjected to mass spectrometry. For acetyltransferase assay, recombinant human KAT2A or KAT8 was incubated with 10 μg calf thymus histones and 10 mM acetyl-CoA in the presence or absence of 10 mM oxaloacetate with or without addition of affinity-purified CS from HepG2 nuclei in a 15 μl reaction mixture containing 50 mM Tris-HCl, pH 8.0, 5% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 5 mM PMSF at 30 °C for 30 min. The reaction materials were then resolved on 15% SDS-PAGE and transferred onto nitrocellulose membranes to detect histone acetylation. Each experiment was performed at least three times.

The sequence of synthesized H3K122 (117-128) succinyl peptide was IRRYQK(succinyl)STELLI. The identity and purity of the peptides were verified by LC–MS. Two micrograms of purified FLAG-SIRT7wt, FLAG-SIRT7H187Y, FLAG-SIRT5 or FLAG-SIRT6 were incubated with 500 ng H3K122succ peptides in desuccinylation assay buffer8 (link) (20 mM Tris·HCl (pH 7.5) and 1 mM DTT) with or without 1.0 mM NAD⁺ in a final volume of 30 μl for 2 h at 37 °C. The reaction mixture was boiled and subjected to dot blot analysis. One microgram of calf thymus bulk histones (Sigma) or mononucleosomes isolated from HeLa cells were incubated with 0.25–5 μg of SIRT7wt, SIRT7H187Y, SIRT5 or SIRT6 in desuccinylation assay buffer in the presence or absence of 1.0 mM NAD⁺ and/or 10 mM NAM in a final volume of 30 μl for 2 h at 37 °C. The reaction mixture was boiled in SDS sample buffer and subjected to SDS–PAGE analysis and mass spectra analysis.