Immunoblotting Analysis of Apoptosis-Related Proteins

Total proteins were extracted from both detached and adherent cells using ice-cold RIPA lysis buffer as previously described [41 (link)]. Protein concentrations were determined using the Bradford assay (500-0006, BioRad, Hercules, CA, USA). Protein levels were analyzed by immunoblotting with antibodies from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France): caspase-9 (9502), cyclin D1 (2878), Bak (3814), Bcl-xL (2764), EGFR (4267) and PARP (9542), as previously described [16 (link)]. GAPDH (FL-335), Mcl-1 (S-19) and β-tubulin (D-10) were from Santa Cruz Biotechnology (Dallas, TX, USA). Bcl-2 (M0887) was from DAKO (Agilent, Santa Clara, CA, USA). Appropriate horseradish-conjugated secondary antibodies (115-035-003 and 111-035-003, Jackson Immunoresearch, West Grove, PA, USA) were used as previously described [16 (link)]. Proteins were detected using enhanced chemiluminescence (170-5061, BioRad Hercules, CA, USA), and signals were visualized with the ChemiDoc MP Imaging System (BioRad, Hercules, CA, USA). Signals were quantified using ImageLab^® software (Biorad, Hercules, CA, USA).