Quantifying DNA Damage Response Signaling

Western blots were performed as reported previously [49 (link)]. In short, cells were pre-treated with 1000 ng/mL cisplatin for 4 hours and irradiated 48 hours later before harvesting at 2 and 24 hours after irradiation. Protein samples were run on polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). Membranes were probed with antibodies against phospho-Chk2 (1:1000, Cell Signaling Technology, Leiden, Netherlands), phospho-p53 (1:1000, Cell Signaling Technology), phospho-ATM (1:1000, R&D, Wiesbaden, Germany), phospho-BRCA1 (1:1000, Cell Signaling Technology) and DNA-PKcs (1:1000, Cell Signaling Technology). β-actin was used as a loading control (1:2000, Cell Signaling Technology). Blots were visualized on X-ray film using a horseradish-peroxidase kit (Cell Signaling Technology).