Anti ku70

SDS-PAGE and western blot analysis were carried out according to the previously published protocol^{33 (link)}. Briefly, cells were collected by trypsinization and were washed in ice-cold PBS. Approximately 3 × 10⁶ cells were lysed in ice-cold RIPA buffer (ThermoFisher Scientific) supplemented with Halt^tm phosphatase and protease inhibitor cocktails (ThermoFisher Scientific), as recommended by the manufacturer and processed for SDS-PAGE and subsequently for western blotting as described^{33 (link)}. The primary antibodies were: anti-RPA32 (mouse hybridoma cell line kindly provided by Dr. J. Hurwitz), anti-KU70 (529) (GeneTex, GTX77607), anti-ATR (Santa Cruz Biotechnology, sc-28901), anti-CHK1 (G-4) (Santa Cruz Biotechnology, sc-8408), anti-pCHK1-S345 (Cell Signaling Technology), anti-DNA-PKcs (Merck Millipore, PC127) and were used at 1:500 to 1:4000 dilutions. The secondary antibodies were anti-mouse IgG conjugated with IRDye680 or anti-rabbit-IgG conjugated with IRDye800 (Li-COR Biosciences, 92668020 and 92632211) at 1:15,000 dilution. Immunoblots were visualized by scanning the membranes in an Odyssey infrared scanner (Li-COR Biosciences). Digital images were processed using the brightness and contrast functions of the dedicated Odyssey software. Raw non-cropped scanned membranes are presented on Fig. S4A–C.

The following antibodies were obtained commercially: anti-γ-tubulin, polyclonal anti-FLAG, anti-Cyclin A, monoclonal anti-FLAG M2, anti-α-tubulin and anti-acetylated-α-tubulin (all from Sigma, St. Louis, MO), anti-Cyclin E, anti-CDK2 phospho-Thr160, anti-Akt and anti-Akt phospho-Thr308 (Cell Signaling, Beverly, MA), anti-centrin 20H5 (Millipore, Billerica, MA), polyclonal anti-β-catenin and anti-GSK3β (Abcam, Cambridge, UK), anti-Ku70 (Genetex, Trvine, CA), anti-DNA-PKcs, and anti-DNA-PKcs phospho-Thr 2609 (Santa Cruz Biotech, Santa Cruz, CA). The immune sera against SF-1 have been described previously [19 (link)].