Western blot analysis of p53, Bax, and Bcl-xl protein levels

The immunoblot assay was used to determine the level of p53, Bax, and Bcl-xl proteins. Briefly, after 24 h culture with various concentrations of methoxy stilbenes or RSV, cell lysates were obtained by extracting HL-60 and THP-1 cells with RIPA buffer. The concentration of total proteins was determined using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Fifty micrograms of total protein from each concentration was resolved by electrophoresis using 12% or 10% SDS-PAGE, followed by electrotransfer onto a polyvinyl difluoride transfer membrane (Immobilon-P, Millipore). After blocking with 10% skimmed milk, the proteins were probed with anti-human p53, Bax, and Bcl-xl antibodies. The β-actin protein was used as an internal control. As the secondary antibodies in the staining reaction, alkaline phosphatase-labeled anti-goat IgG, anti-mouse IgG, or anti-rabbit IgG was used. Bands were visualized with the Bio-Rad AP Conjugate Substrate Kit NBT/BCIP. The amount of the immunoreactive product in each lane was determined using the Quantity One software (Bio-Rad Laboratories). Values were calculated as relative absorbance units (RQ) per mg protein.