Anti chk1 g 4

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 antibodies used were: anti-Chk1 (G-4), anti-Ku86 (C-20), and anti-MyoD (5.8A) from Santa Cruz Biotechnology, Dallas, TX, USA), anti-pSer317-Chk1 (Cell Signaling Technology, Boston, MA, USA), anti-HA (12CA) (Roche Applied Science, Mannheim, Germany), anti-Claspin NT [20 (link)], and anti-E2F (raised in E.W.-F.L. laboratory).

The TRAIP polyclonal antibody was raised against GST-TRAIP-N terminal fusion protein (see Figure 3e) and affinity purified using column coated with MBP-TRAIP-N terminal fusion protein. Antibody specifically recognizing γH2AX was previously described [42 (link)]. The anti-PCNA (PC10) and anti-CHK1 (G-4) antibodies were from Santa Cruz (Dallas, TX, USA); anti-Ki67 antibodies were from Chemicon (Darmstadt, Germany); anti-RPA1 antibodies were from Calbiochem (Darmstadt, Germany); anti-Chk1-pS345 antibodies were from Cell Signaling (Danvers, MA, USA); anti-KAP1 antibodies were from BD Transduction Laboratories (San Jose, CA, USA). Anti-Actin, anti-GFP and anti-Flag (M2) antibodies were obtained from Sigma (Darmstadt, Germany). ATMi (KU55993) and ATRi (VE821) inhibitors were from SelleckChem (Houston, TX, USA).