1mod 1a9

The nuclear (S3 and P3) fractions of 10⁸ MEF or R1 ESCs were used as extracts for immunoprecipitation. Immunoprecipitations were done using antibodies for HP1β (1MOD-1A9, Euromedex) and GFP (#11814460001, Roche; negative control). Protein G-Agarose beads (Roche Applied Science) were washed extensively with wash buffer (30 mM Tris–HCl pH 7.5, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 % Triton X-100, 150 mM NaCl), centrifuged on a 30 % sucrose cushion and washed with 30 mM Tris pH 7.5. The bound proteins were subjected in part to SDS-PAGE silver staining and western blots and analyzed using LC-MS/MS.

Cells were plated on round sterilized 12 mm coverslips in 24-well culture plates (Greiner), coated with gelatin and pre-plated with mitomycin-C treated MEFs. Cells were fixed in 4 % paraformaldehyde for 15 min at room temperature, washed twice with phosphate-buffered saline (PBS) and permeabilized with 0.5 % Triton-X for 5 min at room temperature, washed three times with PBS, and blocked for 30 min with 10 % FCS in PBS at room temperature. Primary antibodies (overnight at 4 °C) included Nanog (R&D, AF2729; 1:20), HP1α (Euromedex, 2HP-1H5-As; 1:750), HP1β (Euromedex, 1MOD-1A9; 1:1750), HP1γ (Euromedex, 2MOD-1G6; 1:750) and H3K9me3 (rabbit polyclonal kindly provided by T. Jenuwein; 1:100). Detection was with anti-rabbit or anti-mouse conjugated to Cy3 or anti-Donkey-FITC (Jackson ImmunoResearch). Images were taken at 60× with an oil NA1.4 lens using a spinning disk confocal microscope (CSUX, Yokogawa, Japan) equipped with an iXon + DU-897-BV monochrome EMCCD camera (Andor, UK) mounted on an Olympus IX81 fully automated microscope, or with an Olympus IX71 epifluorescent microscope equipped with a Dp71 camera (Olympus).

Protein fractionation in the chromatin-bound, the nucleoplasmic or the cytoplasmic compartments was performed essentially as described [81 (link)]. The protein extraction was performed on 4 × 10⁷ primary MEFs (passage 3) or on 4 × 10⁷ R1 mouse ESCs. The protein fractions were separated on 4–20 % gradient Bis-Tris SDS gels (BioRad), blotted, and incubated with the following primary antibodies: HP1β (1MOD-1A9, Euromedex; 1:2000), HP1γ (2MOD-1G6, Euromedex; 1:2000), H3K9me3 (rabbit polyclonal; 1:100), kindly provided by T. Jenuwein (Freiburg), histone H3, kindly provided by M. Bustin (1:10,000, rabbit), and alpha tubulin (#ACLX135B, Accurate Chemical & Scientific Corporation). Other antibodies used for western blots included lamin A/C (sc-20680, SantaCruz; 1:100), hnRNPa2/b1 (ab31645, Abcam; 1:200), phosphoserine (ab9332, Abcam; 1:100) and phosphothreonine (Cell Signaling #93865; 1:3000).