Western Blot Analysis of Checkpoint Proteins

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)].