Mouse monoclonal anti chk1

To evaluate protein expression on Western blot membranes the following antibodies were used: mouse monoclonal anti-pH2AX (Ser 139) (1:100) (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-517348), mouse monoclonal anti-CHK1 (1:100) (clone F-11, Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-8408), mouse monoclonal anti-RAD51 (1:100) (clone G-4, Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-398587), mouse monoclonal anti-p53 (1:100) (clone DO-1, Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-126), rabbit polyclonal anti-p21 (1:200) (clone C-19, Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-397), rabbit polyclonal anti-HSP70 (1:1000) (Proteintech, Rosemont, IL, USA, 10995-1-AP) and rabbit polyclonal anti-HSP90 (1:1000) (Proteintech, Rosemont, IL, USA, 13171-1-AP). Mouse monoclonal anti-β-actin (1:10,000) (Sigma Aldrich, Burlington, MA, USA) or rabbit polyclonal anti-Histone H3 (4499, Cell Signaling, Danvers, MA, USA) were used as loading control. The goat anti-mouse IgG-HRP (1:30,000) (Bethyl Laboratories, Montgomery, TX, USA, A90-116P) and goat anti-rabbit IgG-HRP (1:30,000) (Bethyl Laboratories, Montgomery, TX, USA, A120-101P) were used as secondary antibodies. All the primary and secondary antibodies were diluted in PBS-0.1% Tween20 solution containing 3% of BSA (SERVA).

Whole-cell lysates were prepared as follows: cells were harvested, resuspended in commercial RIPA buffer (Beyotime, P0013B), and then subjected to sonication followed by centrifugation. Western blotting was carried out as previously described [14 (link)]. The primary antibodies used for western blotting were as follows: mouse monoclonal anti-GAPDH (Abmart, 3B3), rabbit polyclonal anti-POT1 (Novus, NB500-176), goat polyclonal anti-ATR (Santa Cruz Biotechnology, sc-1887), rabbit polyclonal anti-phospho-ATR (Abcam, ab178407), mouse monoclonal anti-Chk1 (Santa Cruz Biotechnology, sc-377231), rabbit monoclonal anti-phospho-Chk1 (Cell Signaling Technology, No.2348L). The secondary antibodies used were HRP-conjugated donkey-anti-goat (Santa Cruz Biotechnology, sc2020), fluorescein-conjugated IRDye-680CW goat anti-mouse (LI-COR, 926-32220) and IRDye-800CW goat anti-rabbit (LI-COR, 926-32211).