Salt-extractability of H3K9me3, histone H3, and HP1α in mock cells and RPE cells expressing FLAG-SYCE2. Salt-extractability of H3K9me3, histone H3, and HP1α in mock cells and RPE cells expressing FLAG-SYCE2. (A) Schematic representation of the salt-extractability assay. To prepare whole-cell extracts, cells were washed once in PBS and incubated for 10 min in PBS containing 0.2% NP-40 and protease inhibitors. The soluble fraction and the insoluble nuclear fraction were separated by centrifugation (1,800 g, 10 min, 4°C). The pellet containing insoluble nuclear fraction was then suspended in 0.5 M NaCl-extraction buffer (0.5 M NaCl, 50 mM Tris–HCl [pH 8.0], and 0.05% NP-40). After vortexing intermittently for 1 min (10 s on, 10 s off) and 30 min rotation at 4°C, the supernatant (S1) and pellet were separated by centrifugation (6,500 g, 10 min, 4°C). The pellet was then suspended in 1.5 M NaCl-extraction buffer (1.5 M NaCl, 50 mM Tris–HCl [pH 8.0], and 0.05% NP-40). After vortexing intermittently for 1 min (10 s on, 10 s off) and 30 min rotation at 4°C, the supernatant (S2) and pellet were separated by centrifugation (15,000 g, 10 min, 4°C). The pellet was finally suspended in 2.5 M NaCl-extraction buffer (2.5 M NaCl, 50 mM Tris–HCl [pH 8.0], and 0.05% NP-40). After vortexing intermittently for 1 min (10 s on, 10 s off) and 30 min rotation at 4°C, the supernatant (S3) and pellet (P) were separated by centrifugation (16,000 g, 10 min, 4°C). (B) Each fraction described in (A) derived from mock cells and RPE cells expressing FLAG-SYCE2 was subjected to Western blotting analysis using anti-H3K9me3 antibody, anti-histone H3 antibody, anti-HP1α antibody, and anti-FLAG antibody. Noriko Hosoya et al. LSA 2018;1:e201800021 © 2018 Hosoya et al.