P53 ac k382

Western blot analyses were performed on total cell extracts using the NuPAGE system (Thermo Fisher Scientific) or the Mini PROTEAN TGX gels and the Trans-Blot Turbo Transfer System (Biorad). Antibodies used were: p53-DO7 (Santa Cruz Bio-technology, cat. Sc-47698), TSPYL2 (Bethyl, cat. A304-013A), E2F1, MDM2, cleaved PARP-1 and p53-Ac-K382 (Cell Signalling Technology, respectively cat. 3742 S, 86934 S, 5625 S and 2525 S), γ-H2AX-JBW301, β-actin, GAPDH, FLAG and HA (12CA5) (Merck, respectively cat. 05-6363, A1978, SAB1405848, F1804 and 11583816001). Densitometric analyses were performed with the Fiji software (Schindelin et al. 2012). Original and uncropped western blot images can be found in Supplementary material.

Western blot (WB) analyses were performed with the NuPAGE system (Invitrogen) and densitometric evaluations were done with the ImageQuant 5.2 software (Molecular Dynamics). REGγ phosphorylation analyses were performed using Phos-Tag (Wako) at 30 μM in a 10% acrylamide gel (13 (link)). The antibodies used were: DBC1 (Bethyl Laboratories, A300–434A or Cell Signaling Technology, clone 3G4); p53 (Santa Cruz, DO-7); p53-Ac-K382, phospho-Chk2-T68, PUMA, Cleaved PARP-1 and Cleaved Caspase-3 (Cell Signaling Technology); FLAG (clone M2), β-Actin and SIRT1 (Sigma); HA (clone 12CA5, Roche); REGγ (Transduction Laboratories). Chk2 antibody (clone 44D4/21) was previously described (33 (link)) or purchased from MBL Intl Corp (DCS-270 and DCS-273). Immunoprecipitation (IP) experiments were carried out as described (12 (link)) using cell lysates prepared in ELB buffer (150 mM NaCl, 50 mM Hepes pH 7.5, 5 mM EDTA, 0.5% NP40). After preclearing with protein-G (for mouse antibodies) or protein-A (for rabbit antibodies) coupled sepharose beads (Sigma), proteins of interest were subjected to IP with specific antibodies and analyzed by WB.