Anti γh2a x ab26350 antibody

Degrasyn (WP1130) and PR-619 were purchased from Selleck Chemicals (Houston, TX, USA). FBS, L-glutamine, penicillin and streptomycin solution, 0.25% trypsin EDTA, trypan blue, poly-L-lysine, ribonuclease A, propidium iodide (PI), RIPA buffer, SigmaFAST Protease Inhibitor Cocktail, and TBST buffer were acquired from Sigma-Aldrich (Steinheim, Germany). Annexin V-FITC was obtained from Immunostep (Salamanca, Spain). The CellEvent^® Caspase3/7 Green Flow Cytometry Assay by ThermoFisher (Waltham, MA, USA) was used. Anti-USP5 (sc-390943), anti-USP9X/Y (sc-365353), anti-USP14 (sc-515812), anti-Bcl-2 (sc-7382), and anti-β actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), whereas the anti-Bcl-xl (#2764) antibody was purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA). The anti-γH2A.X (ab26350) antibody was from Abcam (Cambridge, UK), and anti-mouse/HRP and anti-rabbit/HRP antibodies were bought from Dako (Glostrup, Denmark).

For Western blot analysis the cells were seeded in a total of 5 × 10⁶ cells per 25 cm² cell culture flasks and two selected concentration of the compounds were added. After 48 h incubation the cells were harvested, rinsed with cold PBS, suspended in a lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1% NP-40 and protease inhibitors set) and incubated for 20 min on ice. The suspensions were centrifuged at 10,000 rpm at 4 ˚C for 12 min. Then, a sodium dodecyl sulfate (SDS) sample buffer was added to clear supernatants, and the samples were boiled at 95 ˚C for 5 min and subjected to SDS-PAGE on 10–15% gel. For the tests, the resolved proteins were transferred to a PVDF membrane (Millipore, Billerica, MA, USA), using Semidry Transfer Cell (Bio-Rad, Hercules, CA, USA). After the transfer, the membrane was blocked overnight with 1% casein in TBS at 4 ˚C, and then incubated with primary antibody (dilution 1:2000) (Santa Cruz Biotechnology, USA) at room temperature for 1 h, followed by secondary horseradish peroxidase-labelled antibody (Dako, Denmark). The bound antibodies were visualized using ChemiDoc Touch Instruments (BioRad, Hercules, CA, USA). The anti-γH2A.X (ab26350) antibody was from Abcam (Cambridge, UK) while the anti-β actin (C-4) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

BITC was obtained from Sigma-Aldrich (Steinheim, Germany) and dissolved in DMSO (Sigma Aldrich, Steinheim, Germany) to a final concentration of 100 mM immediately prior to the experiments. Histopaque-1077, propidium iodide (PI), ribonuclease A, N-acetyl-l-cysteine (NAC), 2′,7′-Dichlorofluorescin Diacetate (DCF-DA), RIPA buffer and SigmaFAST Protease Inhibitor Cocktail were purchased from Sigma-Aldrich (Steinheim, Germany). Annexin V-FITC was purchased from Immunostep (Salamanca, Spain), CellEvent^®Caspase-3/7 Green Flow Cytometry Assay and an ECL blotting detection system Kit from ThermoFisher (Waltham, MA, USA), CaspGLOW™ Fluorescein Active Caspase-8 Staining Kit from BioVision (Milpitas, CA, USA), and z-VAD-fmk from InvivoGen (San Diego, CA, USA). Anti-γH2A.X (ab26350) antibody was from Abcam (Cambridge, UK), while anti-β actin (C-4) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse/HRP was bought from Dako (Glostrup, Denmark).