P cdc25c ser 216

Primary antibodies CHK1, pCHK1^Ser345, pCHK1^Ser317, CDC25C, pCDC25C^Ser216, Caspase 3 (#9662/#9664 (even mix)), and β‐actin were purchased from Cell Signaling (Beverly, MA, USA). pCDK1^Tyr15 (ab47594) were acquired from Abcam (Cambridge, UK). pH2A.X^Ser139 (#05‐636) was purchased from Millipore. Wee1 (sc‐5285), p53 (sc‐126), and Cyclin A (sc‐751) were obtained from Santa Cruz Biotechnology (Dallas, Tex, USA), whereas CIP2A was brought from Novus Biologicals (Littleton, Co, USA). Cells were harvested and then lysed in ice‐cold NP‐40 lysis buffer as previously described.24 Protein quantification was performed to ensure even loading by Bradford (Bio‐Rad Laboratories AB, Sundbyberg, Sweden) analysis. Proteins (15 μg protein/lane) were separated on Criterion TGX10% Midi Precast Gels in Tris/Glycine Buffers (Bio‐Rad, Hercules CA, USA) at 200 V for 40 minutes, and blotted on PVDF‐membrane using Bio‐Rad Transblot Turbo system according to the manufacturer's instructions. Membranes were blocked in 5% nonfat milk in TBST (20 mmol/L Tris‐Cl, 136 mmol/L NaCl [pH 7.6], 0.1% Tween 20) at room temperature for 1 hour, before they were probed with primary antibodies at 4°C overnight with gentle agitation. Secondary HRP‐conjugated antibodies were visualized using ECL‐plus reagent (GE Healthcare, Chalfont St Gils, UK) by exposure to X‐ray films.

The cells were lysed in Pierce^TM RIPA Buffer (ThermoScientific, Waltham, MA, USA). The protein concentrations were established using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Western blotting was performed using standard procedures, and the blots were developed using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). Original images of western blot added to the Supplementary Data. Primary antibodies used were as follows: ERCC4 (#13465), p-ATM (Ser1981) (#13050), p-ATR (Ser428) (#2853), p-Chk1 (Ser345) (#2341), p-Chk2 (Thr68) (#2197), p-CDC25C (Ser216) (#9528), p-H2AX (Ser139) (#3257000) (1:1000; Cell Signaling Technology, Danvers, MA, USA); NEDD9 (ab18056, 1:1000), beta-Actin (ab49900, 1:100,000) (Abcam; Cambridge, MA, USA), Vinculin (700062) (1:1000, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Vinculin and beta-Actin were used as the loading controls. The quantification was performed using NIH ImageJ Imaging Software (Rayne Rasband, National Institutes of Health, USA).