Immunoblot Analysis of PARP and Caspase-9

Whole cell lysates were prepared as described previously [15 (link), 16 (link)]. Briefly, cells were lysed by suspension in ice-cold lysis buffer [0.1 % sodium dodecyl sulfate (SDS), 1 % NP40, 5 mM ethylene diamine tetraacetic acid (EDTA), 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1 mM sodium orthovanadate, and Complete Protease Inhibitor™ (Roche Applied Science)] and centrifuged at 10,000 × g to pellet cell debris. Approximately 25 μg of each protein sample was then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblot analysis using rabbit polyclonal antibodies specific to PARP [poly (ADP-ribose) polymerase; 1:1,000] or cleaved caspase-9 (Asp315; 1:1,000) (Cell Signaling Technology, Danvers, MA, USA) [17 (link)], β-actin (Sigma-Aldrich), and anti-rabbit IgG horseradish peroxidase-linked secondary antibodies (1:2000 dilution; Cell Signaling Technology). Immunolabeled proteins were then visualized using enhanced chemiluminescence (ECL™) reagents (Amersham Biosciences, Little Chalfont, UK).