Protein Expression Analysis of Apoptotic Regulators

Protein extraction was carried out with approximately 2 × 10⁶ cells treated for 6, 16, 24, and 48 h with OXC or Paclitaxel at the corresponding IC₅₀ using a lysis buffer (20 mM Tris HCl pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, and 10 mM EDTA). The total protein concentration was determined by the Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce), and 25 µg of protein from each sample were subjected to 11% SDS-PAGE polyacrylamide gel electrophoresis using a mini-gel system (mini-Protean II; Bio-Rad Laboratories). Proteins were blotted using polyvinylidene fluoride membranes (PVDF). The transferred membranes were blocked for 1 h with 5% BSA/TTBS (w/v), followed by overnight incubation at 4 °C with the primary antibodies: anti-Bim, anti-Bax, anti-Bcl-2, anti-Bcl-xL, anti-Caspase-7 (GeneTex, Irvine, CA, USA), or anti-Caspase-3 (Thermo Fisher Scientific, Waltham, MA, USA), and anti-α-tubulin (Merck Group, DE, Darmstadt, Germany) was used as a loading control. On the next day, the membranes were incubated with the corresponding secondary antibody, either anti-Mouse IgG or anti-Rabbit IgG (Merck Group, DE, Darmstadt, Germany), for 1 h at room temperature. Bands were visualized on PVDF membranes using the 3,3′-diaminobenzidine tetrahydrochloride (DAB) Substrate Kit detection method (Pierce). ImageJ software was used to semi-quantify and compare band density [5 (link)].