Sc 7150

For the analysis of protein expression, cell pellets were washed and resuspended in PBS. Cells were centrifuged and pellets were stored at −20 °C. Cell pellets were then lysed in Winman’s buffer containing 1% NP-40, 0.1 M Tris–HCl pH 8.0, 0.15 M NaCl, and 5 mM EDTA, complemented with protease inhibitor cocktail (Roche). The total protein content was quantified using the DC™ Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Protein lysate (20 µg) were loaded on 12% SDS-PAGE gel and transferred into a nitrocellulose membrane (GE Healthcare, Cleveland, OH, USA). The following primary antibodies were used: rabbit anti-PARP-1 (1:2000, sc-7150, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-Caspase 3 (1:2000, 05-654, Merck Millipore, Darmstadt, Germany), and goat anti-actin (1:2000, sc-1616, Santa Cruz Biotechnology). The corresponding secondary antibodies were: anti-rabbit IgG-HRP, anti-mouse IgG-HRP, or anti-goat IgG-HRP (1:2000, Santa Cruz Biotechnology). The Amersham™ ECL Western Blotting Detection Reagents (GE Healthcare), the High Performance Chemiluminescence Film (GE Healthcare), and the Kodak GBX developer and fixer (Sigma) were used for signal detection [35 (link),36 (link)].

Total proteins from cell lines were extracted in TNN buffer (50 mM Tris-Cl, pH 7.4; 1% NP-40; 150 mM NaCl; and 1 mM ethylenediaminetetraacetic acid [EDTA]) supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 mM Na₃VO₄) and quantified using the Bradford method. Protein samples (15 μg) were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. After blocking non-specific antibody binding sites, the membrane was incubated overnight at 4 °C with monoclonal antibody against pBRCA1 (9009S, Cell Signaling, Danvers, MA, USA), BRCA1 (sc-6954, Santa Cruz Biotechnology, Paso Robles, CA, USA), cleaved caspase-3 (9664, Cell Signaling), KPNA2 (sc-55537, Santa Cruz Biotechnology), PARP (sc-7150, Santa Cruz Biotechnology), pAMPK (2535S, Cell Signaling), β-actin (A5316, Sigma-Aldrich, St. Louis, MO, USA), and lamin A/C (ab108922, Abcam, Cambridge, MA, USA). All the antibodies were used at a dilution of 1:1000. After incubation with peroxidase-conjugated secondary antibodies at 37 °C for 1 h, the protein bands were visualized using enhanced chemiluminescence reagent (GE Healthcare Biosciences, Piscataway, NJ, USA) and detected using the Amersham Imager 680 (GE Healthcare Biosciences). β-actin and lamin A/C were used for normalization.