Westernbright quantum

We harvested cells at the indicated time points and lysed them in the presence of protease and phosphatase inhibitors. After extraction, proteins were quantified using Bradford assay (Carl Roth). Equal protein amounts were separated by electrophoreses on 4–12% Bis-Tris gradient gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Thermo Fisher Scientific) by electroblotting. We blocked membranes with 5% nonfat dried milk (Carl Roth) or 5% bovine serum albumin (Carl Roth) and incubated them overnight with primary antibody. The next day, we washed the membranes, incubated them with secondary antibody coupled to peroxidase, and washed them again. Protein levels were detected using chemiluminescence (ECL Prime, GE Healthcare or WesternBright Quantum, Biozym).
We used antibodies against p53 (DO-1, sc-126), Mdm2 (SMP14, sc-965), Wip1 (H-300, sc-20712) from Santa Cruz Biotechnology (Dallas, TX), phospho-Chk2 (Thr-68), pSTAT3 (Tyr-705) from Cell Signaling (Danvers, MA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Sigma-Aldrich.

Total cellular protein was extracted by lysis for 30 min on ice in RIPA buffer containing 150 mmol/l NaCl, 1% Triton X-100, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mmol/l EDTA, 50 mmol/l TRIS (pH 7.6), protease inhibitor cocktail (10 μl/ml, Sigma Aldrich), and phosphatase inhibitor (10 μl/ml, Sigma Aldrich). Protein concentrations were determined by bicinchoninic acid protein assay (ThermoFisher Scientific, Darmstadt, Germany). Proteins were separated in SDS-PAGE gels and then wet-blotted to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). Membranes were blocked by 5% non-fat dry milk or BSA in TBS-T (150 mmol/l NaCl, 10 mmol/l TRIS, pH 7.6 and 0.1% TWEEN-20), washed several times, and then incubated with primary antibodies at 4 °C overnight. After several washings with TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Membranes were then developed using Super Signal West Femto (ThermoFisher Scientific) or Western Bright Quantum (Biozym, Hessisch Oldendorf, Germany). α-tubulin was used as a loading control. Antibodies are listed in Additional file 1.