Cleaved caspase 9 asp315

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).

Cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA) following the manufacturer’s instructions. Proteins were quantified using the Qubit 2.0 fluorimeter and the Qubit Protein Assay kit (Thermo Fisher Scientific). Equal amounts of proteins were subjected to gel electrophoresis using the NuPAGE system (Thermo Fisher Scientific). Proteins were transferred to a nitrocellulose membrane, which was blocked with 4% western milk in TBS-TWEEN 0.1% to saturate the non-specific sites. Incubation with primary antibodies was performed overnight at 4 °C, following the manufacturer’s instructions. Antibodies used were phospho-tyrosine (P-Tyr-100) #9411, phospho-SRC family (Tyr416) #2101, SRC family #2108, phospho-AKT (Ser473) #9271, phospho-ERK1/2 (Thr202/Tyr204) #9101, cleaved caspase 3 (Asp175) #9661; cleaved caspase 9 (Asp315) #9505 (Cell Signaling Technology), β-actin #A2066, Vinculin #V9131 (Millipore Sigma). Proteins were visualised with ECL reagent (GE Healthcare, Little Chalfont, UK).