Rabbit anti parp

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.

The whole extracts from OVCAR3 and SKOV3 cells were prepared using RIPA lysis buffer (Promega, Madison, WI, USA). The total proteins concentration in the extracts was quantified by the BCA kit (Beyotime, Shanghai, China). The same amount of each total proteins sample was underwent 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were treated with 5% skimmed milk after transferred to polyvinylidene fluoride (PVDF) membranes. In a 4° C refrigerator, the membranes were probed by primary antibodies (1:1000 dilution) for 12 hour. Rabbit anti-PARP, rabbit anti-c-PARP, and rabbit anti-CLU was provided by Santa Cruz (Dallas, TX, USA). Mouse anti-p-gp and rabbit anti-β-actin was commercially obtained from Cell Signaling Technology (Danvers, MA, USA). The membranes were then treated for 2 hour by horseradish peroxidase-conjugated secondary antibody (1:5000, Solarbio, Beijing, China) at room temperature. Protein blots were exposed by treating with enhanced chemiluminescence (ECL). The gray value of protein blots was quantified by Quantity One software (Bio-Rad, Hercules, CA, USA). In this article, β-actin was the control.