Western blot for the evaluation of Caspase 3 and BCL-2 expression

The protein extraction was performed with RIPA buffer, diluted in Laemmle buffer, separated by electrophoresis using Mini Protean system 3 Electrophoresis Cell (Bio-Rad, Hercules, CA, USA). The proteins were transferred to a nitrocellulose membrane system in the Mini Trans-Blot (Bio-Rad, Hercules, CA, USA). The membrane was incubated with the primary antibody, which was a caspase-3 antibody (Cell SignalingTechnology^®) and BCL-2 (B cell lymphoma -2) overnight. The membrane was washed and incubated with the secondary antibody anti-IgG HRP (Cell SignalingTechnology^®), for 2 h under stirring. Immunodetection was performed using the chemiluminescence according to the manufacturer's instructions (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific, USA). The nitrocellulose membranes were exposed to X-ray films (X-Omat AR film) (Eastman Kodak Co., USA) in predetermined time periods for each protein studied. The antibody used for normalization was GAPDH (6C5) rabbit IgG (Santa Cruz Biotechnology, Inc., Europe) at a 1:5000 dilution. Quantitative analyses of the blots were performed by Scion Image software (Scion Corporation, Frederick, Maryland, USA), which is free software available at http://www.scioncorp.com/.