Phospho atm

Phospho-ATM (Ser 1981, no. AF1655 R&D, 1:250), ATM (no. 2873 Cell Signalling, 1:500), COX-2 (no. 12282 Cell Signalling, 1:250), phospho-p53 (Ser15, no. 9284 Cell Signalling, 1:1,000), p53 (no. 9282 Cell Signalling, 1:1,000) and GAPDH (no. 5174 Cell Signalling, 1:10,000).
Apoptotic cells were detected using ApopTag kit (no. S7100, Millipore). For Sen-β-gal staining43 (link), cells grown on coverslips were fixed in 2% paraformaldehyde made up in PBS for 10 min at room temperature. The cells were washed 2 × 3 min in PBS and then stained at 37 °C for 24 h in Sen-β-Gal staining solution containing 2 mM magnesium chloride, 150 mM sodium chloride, 40 mM citric acid, 12 mM sodium phosphate dibasic, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide and 1 mg ml⁻¹ 5-bromo-4-chloro-3-inolyl-β-D-galactoside (X-Gal) at pH 5.5. The cells were washed 2 × 3 min with PBS, counterstained with DAPI and mounted with vectashield. Epidermal thickness was measured on H&H sections from basement membrane to the start of the stratum corneum, ensuring measurements were taken in interfollicular spaces.

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).