Anti p53 fl393

Western blot analyses were performed as previously described [50 , 51 ]. Briefly, the cells were washed twice with PBS and lysed via sonication in lysis buffer (50 mM Tris-HCl, pH 7.5; 1% NP-40; 1 mM Na3VO4; 150 mM NaCl; 20 mM Na4P2O7; 100 mM NaF; 1% Na-deoxycholate; 0.1% SDS; 1 mM EDTA; phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma)). The samples were separated on 6–15% SDS-PAGE gels, transferred to nitrocellulose membranes (Protran BA83, Whatman) and immunostained. The following primary antibodies and dilutions were used: Anti-p21 [C-19] 1:200 (Santa Cruz, #sc-397), anti-p53 [FL-393] 1:200 (Santa Cruz, #sc-6243), Anti-p16 (M-156) 1:200 (Santa Cruz, #sc-1207), anti-pRb 1:500 (BD-Pharmingen), anti-phospho-pRb (Ser807/811) 1:1000 (Cell Signaling), anti-phospho-histone H2A.X (Ser139) 1:1000 (Millipore 05–636) and monoclonal anti-α-tubulin 1:1000 (Sigma 9026). Horseradish peroxidase-labeled rabbit anti-mouse (Amersham, diluted 1:3000) and goat anti-rabbit (Abcam, #6721, diluted 1:3000) secondary antibodies were used. The proteins were visualized using an ECL detection system (Amersham Biosciences).

Standard western blot assays were used to analyze the levels of protein [14 , 37 (link)]. Anti-p53 (FL393, 1:2 000 dilution, Santa Cruz), anti-MDM2 (2A10, 1:1 000), anti-Flag (F7425,1:10 000 dilution, Sigma), anti-BAG5(ARP61996-P050, 1:1 000 dilution, Aviva Systems Biology, San Diego, CA, USA), anti-HA (sc-7392, 1:2 000 dilution, Santa Cruz), anti-CHIP (sc-66830, 1:1 000 dilution, Santa Cruz), anti-cleaved-Caspase 3 (D175, 1:1 000 dilution, Cell Signaling, Danvers, MA, USA) and anti-β-actin(A5316, 1:20 000 dilution, Sigma) antibodies were used to determine the levels of p53, MDM2, Flag-BAG5, BAG5, HA-BAG5, CHIP, cleaved caspase 3 and β-actin, respectively.

For WCEs, tissues were lysed with RIPA buffer (50 mMTris–Cl, pH 7.5, 150 mMNaCl, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 1 mM EDTA) supplemented with a cocktail of protease inhibitors (Roche, Basilea, CH, Switzerland).
For Western blot, all SDS–PAGE were transferred onto PVDF membranes (Millipore, Burlington, MA, USA). Membranes were developed using the enhanced chemiluminescence (ECL Amersham, Little Chalfont, UK and Cyanagen, Bologna, Italy) by the chemiluminescence imaging system Alliance 2.7 (Uvitec, Cambridge, UK) and quantified by the software Alliance V_1607.
The following primary antibodies were used: anti-p53 FL-393 1:1000 (#sc- 6243 Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-p53 1C12 1:1000 (#2524 Cell Signaling Technology Inc, Danvers, MA, USA), anti-p21 F-5 1:1500 (#sc- 6246 Santa Cruz Biotechnology Inc., Dallas, TX, USA), α-GAPDH 1:8000 (#GA1R, ThermoFisher, Waltham, MA, USA), HRP α-goat 1:8000 (#Sc-2768 Santa Cruz Biotechnology Inc., Dallas, TX, USA), HRP α-mouse 1:5000 (Bio-Rad, Hercules, CA, USA), anti-Bax N-20 1:1000 (#sc-493 Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-Mdm2 mix of clone Ab1 1:1000 (#OP46, Merck KGaA, Darmstadt, Germany) and 2A10 1:1000 for human Mdm2 (#MABE281, Merck KGaA, Darmstadt, Germany).