Affinity-based Protein Isolation and Analysis

Isolation of a nuclear protein fraction and a Triton X-100–insoluble fraction (chromatin-bound fraction), immunoprecipitation, and immunoblot analyses were performed as previously described, with slight modifications [39 (link)]. For nuclear protein extraction, cell pellets were resuspended in MBS-A buffer (10 mM HEPES (pH 7.5), 340 mM sucrose, 1.5 mM MgCl₂, 10 mM KCl, 10%glycerol, 1 mM DTT, 0.1 M PMSF, phosphatase inhibitors and protease inhibitors) and incubated for 8 min at 4°C. After centrifugation, nuclei were further lysed in buffer X (100 mM Tris–HCl, 250 mM NaCl, 1mM EDTA, 1% NP-40, 0.1 M PMSF, phosphatase inhibitors, and protease inhibitors) with Benzonase nuclease followed by sonication and centrifugation. Anti-FLAG M2 agarose (A2220, Sigma), anti-c-Myc agarose (A7470, Sigma), anti-HA agarose (A2095, Sigma), anti-V5 agarose (A7345, Sigma), and anti-S-protein agarose (69704, Novagen) affinity beads were used for immunoprecipitation, as described previously. Agarose beads were incubated with cell lysates for 2 h at 4°C, washed three times with Buffer X at 4°C, and bound proteins were eluted with FLAG-peptide (19–73301, Peptron) or collected by denaturation with 2X SDS loading buffer and resolved by SDS-PAGE and immunoblotting.