Anti patm s1981

A2780, HEC1B and HeLa cells were plated and treated according to the following conditions and as described above for the cell viability or the clonogenic assays. Cells were washed with cold PBS and lysed 1% SDS, 150 mM NaCl, 5 mM NaF, 1% Tween 20, 0.5% NP40, 50 mM Tris–HCl pH 7.5 and 1× Halt protease and phosphatase inhibitor (Pierce). Equivalent amounts of protein lysates were resolved via 4–15% mini-PROTEIN TGX gels (Bio-Rad) and transferred to PVDF membranes. Membranes were blocked for 1 h using 5% non-fat dry milk (Bio-Rad) and incubated with primary antibody overnight at 4 °C, secondary antibody for 4 h at ambient temperature or overnight at 4 °C, and SuperSignal West Dura Chemiluminescent Substrate (ThermoFisher Scientific) for 5 min. Anti-pATM (S1981) and anti-β actin were from Abcam. Anti-ATM was from Thermo Scientific. Anti-ATR, anti-pChk1 (S345), anti-Chk1, anti-pChk2 (Thr68), anti-Chk2, anti-PARP, anti-cleaved PARP, anti-caspase 3, anti-cleaved caspase 3, goat anti-mouse and goat anti-rabbit IgG HRP-linked were from Cell Signaling Technologies. All primary antibodies were used at a 1:1000 dilution. Immunoblots were first probed for cleaved PARP followed by total PARP (Fig. 5B). Similarly, blots were first probed for cleaved caspase 3 followed by total caspase 3 (Fig. 5C and D). Images were acquired using a ChemiDoc XRS+ system (Bio-Rad).