For whole cell extracts cells were washed in PBS and lysed in RIPA buffer (50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (w/v) NP-40, 0,5% (w/v) sodium deoxycholate, 0,1% (w/v) SDS, 10% (v/v) glycerol, 1 mM DTT) for 20 min with rotation at 4°C followed by freeze/thaw lysis in liquid nitrogen. Lysates were cleared by centrifugation at 21,100 g and 4°C for 20 min. The protein content was determined using DC Protein Assay (5000112, Biorad) according to manufacturer’s instructions.

Nuclear extracts were obtained by washing cells in PBS followed by hypotonic lysis in buffer B (10 mM Hepes/KOH pH 7.6, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT) for 15-20 min with rotation at 4°C. Nuclei were pelleted by centrifugation at 4,500 g and 4°C for 15 min. Nuclear proteins were extracted in buffer C (20 mM Hepes/KOH pH 7.6, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM DTT) for 30 min with rotation at 4°C. Extracts were cleared by centrifugation at 21,100 g and 4°C for 45 min. The protein content was determined via Bradford method using Protein Assay (5000006, Biorad) according to manufacturer’s instructions.

Nuclear extract from Drosophila embryos (TRAX) was obtained as previously described [41].

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