Anti caspases 3

After treatment as indicated, cells were collected and lysed using radioimmunoprecipitation assay (RIPA) buffer (Roche, Basel, Switzerland). After protein concentrations were measured using Bradford (Bio-Rad, Madrid, Spain), 40 µg total cellular proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to PVDF membranes. After being blocked, the membrane was probed with specific primary antibodies overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The following primary antibodies were used: anti-PMP22 and anti-caspases-3 were purchased from Abcam (Cambridgeshire, UK), and anti-Bcl-xL was obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-GAPDH and horseradish peroxidase-conjugated second antibody were purchased from Santa Cruz Biotechnology. Bands in the membrane were developed by chemiluminescence method using a chemiluminescence ECL kit (GE Healthcare Biosciences, Pittsburgh, PA, USA), and the relative protein expression levels were determined based on GAPDH expression as a loading control. To further quantify the results, blots were analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).