Quantitative Western Blot Analysis

Cells remaining after the seeding for clonogenic assays were pelleted by centrifugation, lysed in 20 mM Tris HCl pH 7.5, 400 mM NaCl, 5 mM DTT, 20 % glycerol, 0.1 % NP40, 1 mM Pefabloc, Protease Inhibitory Cocktail (Roche), phosSTOP (Roche), 100 nM Ku-0058948, 1 µM ADP-HPD (Trevigen) and analysed by western blot as previously described [11 (link)]. Antibodies used were rabbit anti-PAR (1/1000, 4336-BPC-100, Trevigen), anti-PARG (1/2000, [8 (link)]), anti-actin (1/500, A2066, Sigma), anti-PTEN (1/2000, ab154812, Abcam) and anti-BRCA1 (1/5000, 07-434, Millipore) antibodies. Secondary antibodies were either an Alexa Fluor 680 goat anti-rabbit (1/30,000, Invitrogen) or a peroxidase-coupled goat anti rabbit (1/50,000, Invitrogen), revealed either with Odyssey Infrared Imaging System (Li-Cor, Bioscience) or by chemiluminescence and autoradiography.