Horseradish peroxidase kit

MSCs and HS68 fibroblasts were treated with 400 or 1800 ng/mL bleomycin for 4 hours, and cells were harvested at 24 hours after completion of treatment. Samples containing 10 μg of protein from whole-cell lysates were run on a polyacrylamide gel before transfer to a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Membranes were incubated with a mouse monoclonal antibody against BLMH (1:100, Santa Cruz Biotechnology, Heidelberg, Germany) overnight at 4 °C, and β-actin was used as a loading control (1:2000, Cell Signaling Technology, Leiden, Netherlands). Blots were visualized using a horseradish peroxidase kit (Cell Signaling Technology).

MSCs were treated with 1000 ng/mL cisplatin for 4 hours, and cells were harvested 12 and 24 hours later. Each sample containing 10 μg of total protein from whole-cell lysates was run on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Membranes were probed with primary antibodies against HSP90α (Abcam, Cambridge, UK), HSP90β (Abcam), HSP72 (LifeSpan Biosciences, Eching, Germany), HSP27 (Cell Signaling Technology, Leiden, Netherlands), HSP60 (Cell Signaling Technology), and HSP10 (Santa Cruz, Heidelberg, Germany). β-actin was used as a loading control. Blots were visualized on X-ray film using a horseradish-peroxidase kit (Cell Signaling Technology).

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).