Hybondtm ecltm

Total cell lysates were prepared in RIPA buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1 mM EDTA) added with sodium orthovanadate 3 mM, NaF 100 mM supplemented with protease inhibitors (Sigma-Aldrich). Lysates were kept on ice for 25 min and then centrifuged at 16000 g for 30 min at 4°C. Supernatants were collected and quantified by Bradford protein assay reagent (Biorad). 25 μg of proteins were loaded and separated by SDS-PAGE and electroblotted onto nitrocellulose membrane (Hybond^TM ECL^TM GE Healthcare). Immunoblots probed with the specific antibodies were developed using ECL or ECL Plus chemiluminescence reaction. The following antibodies were used: Snail (L70G2) (Cell Signaling), PARP-1 (clone C2–10 able to detect the 89kDa cleaved fragment of PARP-1; Enzo Life Sciences), PARP-1 (clone F1–23; Enzo Life Sciences), PAR (clone 10HA; Trevigen), Actin (Sigma-Aldrich), Akt (Cell Signaling), Phospho-Akt (Ser473) (Cell Signaling), PTEN (Cell Signaling), Phospho-Histone H2AX (Ser139) (Millipore).

Prior to use, BBMV were centrifuged for 10 min at 16,000 × g and suspended in binding buffer (PBS, 0.1% BSA, pH 7.4). Competition experiments were performed incubating 50 ng of the biotinylated proteins with 20 μg of BBMV in binding buffer. Incubations were carried out for 1 h at 25°C in the absence or presence of an excess of unlabeled proteins (200-fold excess) in a final volume of 100 μl. After incubation, samples were centrifuged at 16,000 × g for 10 min and the pellets were washed with 500 μl of ice-cold binding buffer. The final pellets, containing the bound biotinylated proteins, were suspended in 10 μl of the same buffer and analyzed by 12% SDS-PAGE. The separated proteins were electro-blotted onto a nitrocellulose membrane (HybondTM-ECLTM, GE HealthCare). Biotinylated proteins were visualized after probing with streptavidin-conjugated horseradish peroxidase (1:2000 dilution) with chemiluminescence detection procedure (RPN2109, GE HealthCare) using an ImageQuant LAS4000 image analyzer. Each competition experiment repeated a minimum of three times.