4.7. Western Blot Analysis

Cells were cultured in a 25 cm flask and treated with the IC50 concentration of MA1 24 h before harvest. Cells were then lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, proteases, and phosphatases inhibitors) and centrifuged for 15 min at 13000 rpm in a cold room (5 °C).

Protein concentration was determined by the Bradford test using Bio-Rad protein reagent assay. Then, 30–50 µg of proteins were separated by SDS-PAGE and transferred to a PVDF/nitrocellulose membrane. The membrane was blocked in 5% bovine serum albumin (BSA) in TBS-T and probed overnight with antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) directed against the proteins to be analyzed (e.g., p53, Bcl2, caspase3, caspase9, actin, and GAPDH). Primary antibodies were used at a dilution of 1/200 in the blocking reagent. After a washing step of 3 × 5 min in TBS-T, membranes were incubated at room temperature for two hours with HRP-conjugated secondary IgG antibodies (Thermoscientific, Waltham, MA, USA) at a 1:5000 dilution. Membranes were revealed using ECL Western blotting detection reagent kit (Pierce Biotechnology, Rockford, IL, USA).