Fractionation of U87MG Cells

U87MG cells were fractionated following a previously published protocol ^{51 (link)} with some modifications. Briefly, 5×10⁶ U87MG cells were treated with the plasma membrane lysis buffer (10 mM Tris-HCl, pH 7.5, 0.1% NP-40, 150 mM NaCl) on ice for 4 min. After centrifugation, the supernatant was kept as cytoplasm fraction, the pellet was then treated with nuclei lysis buffer (10 mM HEPES, pH 7.6, 1 mM DTT, 7.5 mM MgCl₂, 0.2 mM EDTA, 0.3 M NaCl, 1 M Urea, 1% NP-40) after washing. The nucleoplasm and chromatin fraction were then separated by centrifugation. Fractionation efficiency was validated by Western Blotting using antibody specific to the marker for each fraction: β-tubulin (Sigma, T8328, 1:2000 dilution) for cytoplasm, rabbit polyclonal U1-70k (a kind gift from Dr. Douglas Black, 1:4000 dilution) for nucleoplasm, and Histone 3 (Abcam, ab1791, 1:2500 dilution) for chromatin.