Anti p atm ser 1981

Whole cell protein extracts were prepared using the SLB buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS). The amount of protein was measured using the BCA method. Equal amounts of proteins (20 µg) were separated electrophorectically in 8%, 12%, or 15% SDS-polyacrylamide gels and afterwards transferred to nitrocellulose membranes. Membranes were blocked in 5% non-fat milk dissolved in TBS containing 0.1% Tween-20 (Sigma Aldrich, Poznan, Poland) for 1 h at room temperature (RT) and incubated with one of the primary monoclonal or polyclonal antibodies: anti-ATM (1:500), anti-p-ATM Ser 1981 (1:1000), anti-DNA-PKcs (1:2000), anti-p-DNA-PKcs Ser 2056 (1:1000), H2AX (1:500) and anti-p-H2AX Ser 139 (1:1000) (Abcam, Cambridge, UK), anti-p53 (DO-1) (1:500), anti-p21(C-19) (1:500) (Santa Cruz Biotechnology Inc., Dallas, Texas, USA), anti-p-p53 Ser 15, anti-Chk2, anti-p-Chk2 Thr 68, anti-p65 and anti-p-p65 Ser 536 (Cell Signaling, Lab-JOT Ltd., Warsaw, Poland), anti-PARP1 (1:1000) (Becton Dickinson, Diag-med, Warsaw, Poland), and anti-GAPDH (1:50000) (Merck Millipore, Warsaw, Poland). The proteins were detected with appropriate secondary antibodies conjugated with horseradish peroxidase and ECL reagents (Thermo Scientific), according to the manufacturer’s protocol.

Whole cell protein extracts were prepared according to the Laemmli method [13] . Equal amounts of protein were separated electrophorectically in 8, 12 or 15% SDS-polyacrylamide gels and afterwards transferred to nitrocellulose membranes. Membranes were blocked in 5% non-fat milk dissolved in TBS containing 0,1% Tween-20 (Sigma Aldrich, Poznan, Poland) for 1 h at RT and incubated with one of the primary monoclonal or polyclonal antibodies: anti-ATM (1∶500) (Millipore, Merck, Warsaw, Poland), anti-p-ATM Ser 1981 (1∶1000), H2AX (1∶500) and anti-γH2AX (1∶1000) (Abcam, Cambridge, UK), anti-p16 (1∶500), anti-p53 (DO-1) (1∶500), anti-p21 (C-19) (1∶500) (Santa Cruz Biotechnology Inc., Dallas, Texas, USA), anti-p-p53 Ser 15, anti-Chk1, anti-p-Chk1 Ser 317, anti-Chk2, anti-p-Chk2 Thr 68, anti-NBS1 Ser 343 (Cell Signaling, Lab-JOT Ltd., Warsaw, Poland), anti-PARP1 (1∶1000) (Becton Dickinson, Diag-med, Warsaw, Poland) anti-NBS1 (1∶500), anti-β-actin (1∶50000) (Sigma Aldrich, Poznan, Poland) and anti-GAPDH (1∶50000) (Millipore, Merck, Warsaw, Poland). The proteins were detected with appropriate secondary antibodies conjugated with horseradish peroxidase and ECL reagents (GE Healthcare, Buckinghamshire, UK), according to the manufacturer’s protocol.