Nuclear extraction and Co-ImmunoPrecipitation

Nuclear extraction was performed using standard protocols. Shortly, cells were harvested by washing in ice-cold PBS and collected by centrifugation (300 g at 4°C). Cell lysis was performed in hygroscopic conditions using V1 buffer (10mM HEPES-KOH pH 7.9; 1.5mM MgCl2; 10mM KCl and 1μM dexamethasone, 0.5mM DTT, 0.15% NP40, protease inhibitors and PhosphoSTOP) while douncing on ice. Crude nuclei were collected by centrifugation (2,700 g at 4°C) and nuclear lysis was performed in V2 buffer (420mM NaCl; 20mM HEPES-KOH pH 7.9; 20% glycerol; 2mM MgCl2; 0.2mM EDTA and 1μM dexamethasone; 0.5mM DTT; 0.1% NP40; protease inhibitors and PhosphoSTOP) while incubating for 1h at 4°C and subsequent centrifugation at 21,000 g (4°C). Supernatants were directly processed for co-IP. Co-IP was performed by diluting 200 μg 1:1 in AM100 (100mM KCl, 5mM MgCl2, 20mM Tris (pH 8.0), 0.2mM EDTA and 20% glycerol) with EDTA-free proteinase inhibitors and pre-cleared with pre-blocked sheep α-rabbit or sheep α-mouse IgG Dynabeads for 2h at 4°C while agitating. IPs were incubated with 1μg of antibody (Key resources table) for 2h at 4°C. Subsequently, pre-blocked sheep α-rabbit or sheep α-mouse IgG Dynabeads (ThermoFisher Scientific) were collected after 3 washes with buffer AM100 plus 0.5% Triton. Bound proteins were eluted in Laemmli buffer and analyzed by western blots. For Co-IP from whole cell lysates, RAW264.7 cells were lysed in NP40 buffer (25mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1% NP40, 5% glycerol, freshly added phosphatase and proteinase inhibitors), sonicated for 15 s and cleared by spinning 10min at 12,000xg. After preclearing samples were diluted 1:5 in Tris IP buffer (20mM Tris pH 7.5, 2mM MgCl₂, 100mM NaCl, 0.2mM EDTA, 0.1% Triton X-100, freshly added phosphatase and proteinase inhibitors) for incubation with 1ug of primary antibody (Key resources table) overnight at 4°C. Pre-blocked beads were added for 3h on the next day and proteins eluted from the beads after three washes with Tris-IP buffer, one wash with Tris-IP buffer including 500nM NaCl and 1 wash of Tris-IP buffer including 500nM NaCl and 1% Triton X-100.