Nucleic Acid Extraction and Western Blot

The assay was performed as previously described^{31 (link)}. Treated cells were lysed with MB buffer (6 M guanidinium isothiocyanate, 10 mM Tris-HCl, pH 6.5, 20 mM EDTA, 4% Triton X100, 1% Sarkosyl, and 1% dithiothreitol). Nucleic acids were precipitated by adding 100% ethanol to the cell lysate to a final concentration of 33% (1/2 volume of the cell lysate). After 5 min of incubation at −20 °C, nucleic acids were collected by centrifugation. Pellet was washed with 75% ethanol and dissolved in freshly made 8 mM NaOH solution. DNA concentration was determined by NanoDrop 1000 spectrophotometer. 1 µg DNA from each sample was transferred to a nitrocellulose membrane using the Bio-Dot SF microfiltration apparatus (Bio-Rad, #170-6542). The membrane was blocked in 5% non-fat milk in TBS with 0.1% Tween-20, then probed with primary antibodies and horseradish-peroxidase-conjugated secondary antibodies. The signal was visualized by Western Lightning Plus-ECL (PerkinElmer). Antibodies used were listed in section ‘Western blotting and band depletion assays’.