Mouse monoclonal pcna

Cell lysates were prepared in radioim-munoprecipitation assay buffer [20 mmol/l Tris-HCl (pH 7.5), 0.1% (w/v) sodium lauryl sulfate, 0.5% (w/v) sodium deoxycholate, 135 mmol/l NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mmol/l EDTA] supplemented with Complete protease inhibitor cocktail (Roche Molecular Biochemicals) and phosphatase inhibitors sodium fluoride (20 mmol/l) and sodium vanadate (0.27 mmol/l). Western blot analysis was performed as previously described (20 (link)). The total protein concentration was determined using the protein assay dye reagent from Bio-Rad Laboratories. Cell lysates in SDS-sample buffer were boiled for 5 min and equal amounts of total protein were analyzed by 10% SDS-PAGE and western blotting. The antibodies used in this study are mouse monoclonal p53 (1:250) from Santa Cruz Biotechnology; rabbit polyclonal cleaved caspase-3 (1:1,000), rabbit polyclonal cleaved PARP (1:1,000), mouse monoclonal GAPDH (1:1,000), and mouse monoclonal PCNA (1:1000) from Cell Signaling Technology. Proteins were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NB, USA) and analyzed with Licor Image Studio 2.0 acquisition and analysis software.