Co-immunoprecipitation

For co-immunoprecipitation assays, SW480 cells were treated with 12.5 ng ml⁻¹ TNF-α for 0 or 16 h, crosslinked in 1% formaldehyde (Sigma), harvested, and washed with PBS. The cell pellet was resuspended in RIPA lysis buffer (50 mM Tris-HCl at pH 7.9, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% Na-deoxycholate), supplemented with protease inhibitor cocktail (Sigma) and incubated on ice for 30 min before isolating the nuclear pellet by centrifugation at 4 °C. The pellet was then further lysed by resuspension in hypotonic buffer (20 mM Hepes at pH 7.9, 1.5 mM MgCl₂, 20 mM KCl, 25% glycerol) and high-salt buffer (20 mM Hepes at pH 7.9, 1.5 mM MgCl₂, 800 mM KCl, 25% glycerol, 1% NP40) followed by rotation at 4 °C. Lysates were cleared by centrifugation and incubated with indicated antibodies for 2 h at 4 °C. After an additional 2 h incubation with Protein A Sepharose (Rockland Inc.), beads were washed with wash buffer (20 mM Tris-HCl at pH 7.9, 20% glycerol, 0.1 mM EDTA, 150 mM KCl, 0.1% NP40) five times and analyzed by immunoblotting.