Anti cleaved parp 1 antibody

Frozen lung tissue was thawed on ice and whole lung homogenates were prepared using a tissue homogenizer in RIPA buffer containing the protease inhibitor cocktail. Homogenates were centrifuged at 17,000 g for 15 min at 4°C, and total protein concentrations were determined using the Bradford assay (BioRad, Hercules, CA). A total of 50 μg protein of each sample was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on 4-12% NuPage Bis-Tris gradient gels (Invitrogen) and transferred onto nitrocellulose membranes at 35 mV for 2 hours. Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline (Bio-Rad) containing 0.1% Tween-20 (TBST) for 1 h at 37°C. The membranes were then incubated overnight with an anti-cleaved PARP-1 antibody (Cell Signaling, 1:1000) or an anti-total PARP-1 antibody (Cell Signaling, 1:1000) at 4°C. The next day, after 3 washing steps in TBST, all membranes were incubated for 1 hour at room temperature with an anti-rabbit HRP-conjugated IgG antibody (1:3000, Cell Signaling). Bands were visualized by enhanced chemoluminescence with ECL SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Rockford IL). Band densitometry measurements to determine relative quantities of protein were performed using ImageJ 1.42 software for Windows.