Subcellular fractionation

Cells were lysed in NP-40 lysis buffer (l0 mM Tris, pH 7.9, 140 mM KCl, 5 mM MgCl₂and 0.5 % NP-40) supplemented with protease and phosphatase inhibitors at a concentration of 3-5x10⁷ cells/ml. Cell were gently mixed and kept on ice for 15 min. The lysates were then centrifugated at 1000 x g for 5 min. The supernatant represents the cytosolic plus the membrane fraction. The pellets (nuclear fraction) were washed twice in NP-40 lysis buffer and then sonicated in Laemmli sample on ice using a Misonix sonicator Processor XL2020 equipped with a horn/probe (fine tip). The sonication processing time was 1 min with pulses of 10 sec every 5 sec intervals and power setting at position 2.