Antiphospho chk1 s345

Protein extracts (30 µg/lane) were electrophoresed and electroblotted as previously described [18 (link)]. Electroblotted membranes were blocked with 5% non-fat milk (Nestlé Carnation, Solon, OH, USA) in TBS (Cell Signaling Technology, Boston, MA, USA) for 60 min at room temperature. They were then incubated with either primary 1:1000 antiphospho-Chk1[S345] (Cell Signaling Technology, #2348), 1:1000 anti-Chk1 (Cell Signaling Technology; #2360), 1:1000 anti-phospho-Chk2[T68] (Cell Signaling Technology; #2197), 1:1000 anti-Chk2 (Cell Signaling Technology; #6334), 1:500 anti-mutant p53 (abcam, Boston, MA, USA; #ab32049), 1:1000 anti-p53 (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-6243), or 1:5000 anti-β-actin (Santa Cruz Biotechnology; #sc-47778) overnight at 4°C, followed by the corresponding horseradish peroxidase-conjugated goat anti-rabbit (Cell Signaling Technology; #7074) or anti-mouse (Cell Signaling Technology; #7076) secondary IgG for 2 h at room temperature in TBST (Cell Signaling Technology) with 5% non-fat milk (Nestlé Carnation) or 5% BSA (Cell Signaling Technology), according to the antibody manufacturer’s recommendations. Washed membranes were developed for optical density quantitation of chemifluorescent signals from replicate immunoblots as previously described [18 (link),24 (link)].

Cell lysates were prepared in a buffer consisting of 20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25mM sodium pyrophosphate, 1 mM NaF, 1mM b-glycerophosphate, 0.1 mM sodium orthovanadate, 1mM PMSF, 2mg/ml leupeptin and 10 mg/ml aprotinin. Cell lysates containing 50mg protein were separated on SDS-PAGE gel, and transferred onto Hybond ECL nitrocellulose membranes (Amersham), followed by treatment with 5% skim milk at room temperature for one hour as well as incubation with individual primary and secondary antibodies. Signals were then developed (ECL Western Blotting Kit, Amersham). Primary antibodies used were: monoclonal anti-BMI1 (1:1000, Invitrogen), anti-H2AX (1:1000, Millpore), anti-γH2AX (1:1000, Cell Signaling), anti-T1989 phosphorylated ATR (1:1000, Abcam), anti-ATR (1:500, Santa Cruz), anti- phospho-CHK1 (S345) (1:500, Cell Signaling), anti-CHK1 (1:1000, Cell signaling), anti-TOPBP1 (1:500, Bethyl), anti-phospho-CDK1 (Y15) (1:1000, Cell Signaling), anti-CDK1 (1:1000, Santa Cruz), anti-tubulin (1:1000, Santa Cruz), and anti-actin (1:1000, Santa Cruz).