β tubulin d 10

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).

Protein levels of both detached and adherent cells were analyzed by immunoblotting with PARP (9542), Bcl-xL (2764) and Bak (3814) antibodies from Cell Signaling Technology (Ozyme, Saint-Quentin-en-Yvelines, France), with Mcl-1 (S-19), GAPDH (FL-335) and β-Tubulin (D-10) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA), and with the Bcl-2 (M0887) antibody from DAKO (Agilent, Santa Clara, CA, USA) as previously described (26 (link), 27 (link)). Each immunoblot is representative of at least three distinct experiments. Protein expression was estimated by quantifying the density of immunoblots bands adjusted to GAPDH or β-Tubulin (ImageLab® software).