Anti p chk1 ser317

The following antibodies were used: anti-RNase H2C (ProteinTech, 1 : 1000), anti-RNase H2A (Abcam, 1 : 1000), anti-Actin (Santa Cruz Biotechnology, 1 : 10 000); anti-RNase H2B (produced in this work, 1 : 500); anti-Caspase3 (Cell Signaling, 1 : 1000); anti-PCNA (PC-10, 1 : 200) was kindly provided by S. Sabbioneda; anti-BrdU FITC-conjugated (BD Biosciences, 1 : 50), anti-Chk1 (Cell Signaling, 1 : 200); anti-Chk2 (Cell Signaling, 1 : 1000); anti-P-Chk1-Ser317 (Cell Signaling, 1 : 1000); anti-P-Chk2-Thr68 (Cell Signaling, 1 : 500); anti p53 (DO1, GeneSpin, 1 : 1000); anti-P-p53-Ser15 (Cell Signaling, 1 : 1000); anti-53BP1 (Cell Signaling, 1 : 150 for immunofluorescence; 1 : 1000 for western blot); anti-Vinculin (Sigma, 1 : 40 000); anti-Vimentin (Cell Signaling, 1:10 000); anti-Tubulin (Sigma, 1 : 1000). Secondary antibodies were goat anti-mouse or goat anti-rabbit conjugated to HRP (western blot) or to Alexa Fluor 488 or Alexa Fluor 594 (immunofluorescence). Hydroxyurea was used at a final concentration of 0.1 mm. Puromycin was used to a final concentration of 1 μm.

The antibodies used in this study were the following: anti-thymine dimer (Insight Biotechnology), (anti-γH2AX, MABE205, Millipore, Schwalbach, Germany), anti-pATM (ser1981, Rockland), anti-ATR (Santa Cruz), anti-pATR (ser428, Cell Signalling), anti-Chk1 (Santa Cruz), anti-pChk1 (ser317, Cell Signalling), and anti-tubulin (YL1/2, Abcam), FLAG-M5 monoclonal antibody (A2220, Sigma), anti-p300 (C-20, Santa Cruz), anti-tubulin (YL1/2, Abcam), anti-MAML1 (Cell Signalling), anti-SMAD3 (Abcam).

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.