Mouse anti bcl 2

Cells were infected and/or treated and cell lysates prepared after 48 h, in RIPA buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 1% (v/v) NP40 and 0.1% (w/v) SDS) containing phosphatase and protease inhibitors (PhosphoSTOP; Roche Diagnostics, Switzerland). Proteins were quantified using the Bio-Rad Protein assay (Bio-Rad), prepared in sample Laemmli buffer (0.125 m Tris-HCl pH6.8, 20% glycerol, 4% SDS, 0.01% bromophenol blue, 10% β-mercaptoethanol) and 15–20 μg of total protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, transferred to polyvinylidene difluoride membranes (Merck-Millipore, MA) and identified with the following antibodies: rabbit anti-LC3B (1:1000), goat anti-Ad hexon (1:1000) and mouse anti-vinculin (1:5000) (Abcam, UK), mouse anti-Bcl-2 (1:1000; Dako, UK), mouse anti-AdE1A (1:2000; GeneTex, TX), rabbit anti-Atg5, rabbit anti-PARP and mouse anti-p62 (1:1000; Santa Cruz Biotechnology, CA) and goat anti-actin, goat anti-Ku-70 (1:5000; Santa Cruz Biotechnology) and rabbit anti-Atg7 (1:2000; Merck-Millipore). Detection was by horseradish peroxidase-conjugated secondary antibodies (Dako) and the enhanced chemiluminescence reagent Plus-ECL (PerkinElmer, MA), visualised by autoradiography X-Ray film or digital capture (Chemidoc; GE Healthcare, UK). Densitometric analysis was performed using the NIH ImageJ software.