Western Blot Analysis of DNA Damage Response

Cells were harvested and centrifuged at 5000 rpm for 5 min at 4 °C. The supernatant was removed, Buffer WCE 2X (100 mM TrisHCl pH 6.8, 4% SDS, 200 mM DTT) was added to resuspend the cell pellet, boiled for 10 min and then added an equal volume of SDS-PAGE Sample Loading Buffer [2X] (100 mM TrisHCl pH 6.8, 4% SDS, 200 mM DTT, 20% glycerol, 0.004% bromphenol blue) to the mixture. Cell extracts were pelleted at 15,000 g in an Eppendorf centrifuge for 15 min at 4 °C and the supernatants were analyzed by Western blotting according to [55 (link)], using the following antibodies, all diluted in TBS-T: anti-p-p53 (Ser 15; 1:1000, Santa Cell Signaling), anti-p53 (1:1000, Santa Cruz), anti-p-H2AX (Ser 139; 1:1000, Millipore), anti-SUV39H1 (44.1; 1:1000, Santa Cruz Biotechnology), anti-TDP-43 (1:5000, Proteintech), anti-H3k9me3 (1:1000, Abcam ab8898), anti-H3K9me2 (1:500, Abcam ab1220), anti-actin-HRP-conjugated (1:5000, Santa Cruz Biotechnology). These primary antibodies were detected using HRP conjugated anti-mouse and anti-rabbit IgGs and the ECL detection kit (all from GE Healthcare). Band intensities were quantified by densitometric analysis with Image Lab software (Bio-Rad).
All full length uncropped original western blots are available in the Supplementary Materials section.