Whole Cell Protein Extraction and Analysis

Whole cell extracts were obtained by lysing cells in 1% SDS and 10 mM Tris-HCl (pH 7.4) supplemented with protease inhibitors (Sigma-Aldrich) and phosphatase inhibitors (Halt phosphatase inhibitor cocktail; ThermoFisher). Viscosity of the samples was reduced by brief sonication. To detect PAR, cell extracts were performed as described previously^{31 (link)}. To detect ATM, ATM-pS1981, DNA-PK and DNA-PK-pS2056, cell extracts were prepared as previously described^{22 (link)}. Proteins were separated by SDS-PAGE and immunoblotted with the following antibodies: anti-actin (MAB1501; Millipore), anti-ATM (ab32420; Abcam), anti-ATM-pS1981 (ab81292; Abcam), anti-cleaved caspase-3 (#9664; Cell Signaling), anti-caspase-9 (#9502; Cell Signaling), anti-Chk1 (sc84081; SantaCruz), anti-Chk1-pS345 (#2348; Cell Signaling), anti-DNA-PK (ab1832; Abcam), anti-DNA-PK-pS2056 (ab18192; Abcam), anti-HIF1α (NB100–449; Novus), anti-PAR (AM80–100UG; Millipore), anti-p53 (#48818; Cell Signaling), anti-p53-pS15 (#9284, Cell Signaling), anti-PARP-1 (#9542; Cell Signaling), anti-TOP1 (ab109374; Abcam), anti-Tubulin (T5168; Sigma-Aldrich), anti-V5 tag (46–0705; Invitrogen). Immunoblotting was revealed by chemiluminescence using ChemiDoc MP System (Bio-Rad). Quantification of protein levels was done with Image Lab software (version 4.1).