P53phosphos15

Cells were harvested in protein lysis buffer (20 mM TRIS-HCl pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1 mM beta-glycerophosphate, 2 M Urea, Protease inhibitor Cocktail, Roche). After 10 min lysis on ice, samples were briefly sonicated to disrupt DNA-protein complexes. Total protein concentration was measured using a Pierce BCA Protein assay kit (Thermo Scientific Fisher). After boiling the samples in Laemmli buffer at 95°C for 5 min, equal amounts of protein samples were separated by SDS-PAGE, transferred onto nitrocellulose, and visualized with the following antibodies: PARP1 (9542, Cell Signalling), γH2AX (S139) (9718, Cell Signalling), β-Actin (ab8227 abcam), p21 (2947, Cell signalling), Mdm2 (OP 46, Calbiochem), p53K382Ac (252S, Cell Signalling), p53phosphoS15 (9287S, Cell Signalling), p53 (DO-1 sc-126, Santa Cruz), phospho Chk2 (C13C1 2197, Cell Signalling).

Antibodies were as follows: p53 (DO-1; Moravian Biotechnology), p21 ((H-164): sc-756; SantaCruz Biotechnology), PLK1 (208G4; Cell Signaling Technology), actin (A2066; Sigma), Poly (ADP-Ribose) Polymerase-1 (PARP) (9542; Cell Signaling Technology), γ-H2AX (Phospho-S139) (ab11174; AbCam), p53 (Phospho-S15) (#9284; Cell Signaling Technology), MDM2 (4B2; Moravian Biotechnology), Gamma-tubulin (T65557; Sigma), Histone H3 ((D1H2): 4499; Cell Signaling Technology), Histone H3 (Phospho-S10) (06–570; Millipore), BrdU Pure ((B44): 347580; Becton Dickinson).
PLK1 inhibitors, GSK461364 and BI6727, ATM and ATR inhibitors, KU-55933 and VE-821, and etoposide were from Selleckchem. The specificity and efficacy of GSK461364 and BI6727 have been described previously^{36 (link),37 (link)}. S-Trityl-L-cysteine (STLC) was from Sigma. Taxol was from LC Labs. Nocodazole was from Millipore.