2.6. Western Blot Analysis

The signaling pathways of MCF-7 cell apoptosis induced by C. militaris concentrate and cordycepin were evaluated using western blot analysis. Briefly, cells at a density of 4 × 10⁵ cells/3 mL were seeded onto 6-well plates. After incubation for 24 h, C. militaris concentrate and cordycepin at various concentrations were added. After treatment for 24 h, cells were collected using a cell scraper and lysed using radio immunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, Inc., MA, USA) containing 1× ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). The protein concentrations of samples were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Carlsbad, CA, USA). Protein (20 μP) was mixed with 4× NuPAGE LDS Sample Buffer (Thermo Scientific, Carlsbad, CA, USA). After boiling for 10 min, the proteins were separated on precast 4–15% Mini-PROTEAN TGX (Tris-Glycine. eXtended) gel (Bio-Rad, Hercules, CA, USA) and then electrotransferred onto polyvinylidene fluoride (PVDF) transfer membranes. To enhance the detection of proteins, membranes were incubated with specific primary antibodies to Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cleaved caspase-7, cleaved caspase-8, X-linked inhibitor of apoptosis protein (XIAP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Inc.), followed by incubation with horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (Cell Signaling Technology, Inc.). The bound antibodies were visualized using Pierce ECL (enhanced chemiluminescence) Western Blotting Substrate (Rockford, IL, USA) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).