Rabbit monoclonal anti p53

The following antibodies were used: mouse monoclonal anti-PARP1 antibody (BD Bioscience, San Jose, CA, USA), mouse monoclonal anti-HIPK2 antibody^{46 (link)} (kindly provided by Dr H Koseki, RIKEN Research Center, Yokohama, Japan), rabbit polyclonal anti-PAR (BD Bioscience), mouse monoclonal anti-α-tubulin (Sigma-Aldrich), mouse monoclonal antibodies for Myc, HA, Flag and GST (abm, Richmond, Canada), rabbit polyclonal anti-phospho-p53 (serine 46) (BD Bioscience), mouse monoclonal anti-ubiquitin (Millipore, Billerica, MA, USA), rabbit monoclonal anti-p53 (Cell Signaling, Danvers, MA, USA), rabbit polyclonal anti-HSP70 (Stressgen, San Diego, CA, USA), rabbit polyclonal anti-CHIP (kindly provided by Dr S Yoo, Kyung Hee University, Korea). Specificity of the antibody that reacts with the endogenous HIPK2 protein was confirmed by detecting the accumulated endogenous HIPK2 following DNA damage and the exogenously expressed HIPK2 (Supplementary Figure S3).

Cells were plated in a 6-well plate as described above. Cells were lysed on plates in TNN buffer. After centrifugation (5 minutes, 800rpm) the supernatants containing the isolated proteins was kept at −80°C. Protein concentrations were determined using the Pierce^® BCA protein assay kit (ThermoScientific). Western blot analysis was performed as described previously [35 (link)].
Following antibodies were used: rabbit monoclonal anti-p53 (1:2000, Cell Signaling Technology, Leiden, the Netherlands, no. 9282); mouse monoclonal anti-MDM2 (3G9) (1:1000, Millipore, Overijse, Belgium, no. 04-1555), rabbit monoclonal anti-p21 (1:2000, Abcam, Cambridge, UK, no. ab109199), rabbit monoclonal anti-PUMA (1:2000, Abcam no. ab33906) and rabbit monoclonal anti-BAX (1:2000, Abcam no. ab32503). Mouse monoclonal anti-β-actin was used as internal standard (1:5000, Sigma Aldrich, Diegem, Belgium). Anti-mouse and anti-rabbit HRP-labeled secondary antibodies were used (1:2000, Cell Signaling no. 7076S and no. 7074S) and chemiluminescent detection was performed using the WesternBright^TM Quantum Western blotting detection kit (Advansta, Temse, Belgium).

Whole-cell lysates were obtained using the ProteoJET Mammalian Cell Lysis Reagent (Thermo Scientific, Rockford, IL, USA). Proteins (50 μg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dried milk in TBST (50 mM Tris (pH 7.5), 200 mM NaCl, 0.05% Tween 20) at room temperature for 1 h. The membranes were then incubated with the primary antibody in the blocking solution for 1 h at room temperature, washed three times with TBST for 15 min, incubated with the HRP-conjugated secondary antibody at room temperature for 1 h and then washed three times with TBST. Antigen-antibody complexes were detected using the SuperSignal detection reagents (Thermo Scientific, Rockford, IL, USA). The following antibodies were used: rabbit monoclonal anti-p53, rabbit monoclonal anti-AGO2, rabbit monoclonal anti-GADD45A, mouse monoclonal anti-GAPDH (Cell Signalling Technology, MA, USA) and horseradish peroxidase-conjugated goat-anti-rabbit and goat-anti-mouse secondary antibodies (Santa Cruz. CA, USA).