4.10. Western Blot Analysis of PARP-1, p53, p73, Bcl-2, Bax and NME1/2 Protein Expression

Following treatment, the whole-cell lysates were prepared by scraping cells into PBS with protease inhibitors (Complete, Mini, EDTA-free protease inhibitor cocktail tablets; Roche, Indianapolis, IN, USA) and sonicated (1 mm probe, 2 × 15 s). The protein concentration was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Extracted proteins (50 μg) were resolved using SDS–PAGE and analyzed by immunoblotting on nitrocellulose membranes (Whatman, GE Healthcare, Life Sciences, Berlin, Germany). Nonspecific binding was blocked by 5% nonfat milk in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 30 min at RT. Immunoblots were incubated overnight with primary antibody and then for 1 h at RT by appropriate secondary antibody. Blots were incubated with primary anti-PARP-1 antibody (F-2: sc-8007; Santa Cruz Biotechnology, 1:1000), anti-p53α antibody (TSRα kindly provided by Prof. J-C. Bourdon, Dundee, United Kingdom; 1:2000), anti-p73 antibody (ab40658, Abcam, 1:3000), anti-Bcl-2 antibody (C-2: sc-7382; Santa Cruz Biotechnology, 1:1000), anti-Bax antibody (B-9: sc-7480; Santa Cruz Biotechnology; 1:500) and anti-NME1/NME2 antibody (kindly provided by I. Lascu, Bordeaux, France and S. Volarević, Rijeka, Croatia; 1:3000). β-actin (7D2C10, 60008-1-Ig, Proteintech; 1:10,000) was used for normalization. Immunoreactive bands were visualized by chemiluminescence detection (Western Lightning Plus-ECL Enhanced Chemiluminescence Substrate, PerkinElmer, Waltham, MA, USA) and quantified by using ImageJ NIH software after detection with Alliance 4.7 (UVItec Cambridge, London, UK).