Nuclear extracts

5 × 10⁶ THP-1 cells were pelleted at 500 × g for 5 min at 4°C in a 15 ml conical tube. The pellet was resuspended and washed twice with PBS. Cell pellet was resuspended in 1 ml of ‘low-salt buffer’ (10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl plus 1 μl protease inhibitor cocktail (Sigma-Aldrich, cat # P8340) and incubated for 10 min on ice. 50 μl of 5% IGEPAL (Sigma-Aldrich, cat # I8896) was added to the cell suspension and vortexed for 10 seconds. Released nuclei were pelleted at 750 × g for 5 min at 4°C. The supernatant was saved as the ‘cytosolic fraction’. To wash the remaining cytosolic proteins from the surface of the nuclear pellet, 100 μl of the low-salt buffer was gently pipetted onto the side of the tube and allowed to wash the pellet, making sure to not disrupt the pellet. This wash was then gently transferred to the cytosolic fraction without dislodging the nuclear pellet. 200 μl of ‘high-salt buffer’ (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 420 mM NaCl plus 1 μl protease inhibitor cocktail) was pipetted on the pellet and the tube went through a vigorous vortex for 30 s followed by nutation at 4°C for 1 h. The nuclei were pelleted at 4°C for 20 min at 21 000 × g. The supernatant was transferred into another tube as the nuclear soluble protein fraction. Final nuclear extract samples used in nextPBM assays were 9.6 mg/ml.