Western Blot Analysis of Apoptosis Regulators

Total cell lysates were obtained with an extraction buffer as previously described. Protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). For Western blot analysis, the cell lysates were separated by 12% SDS-PAGE, transferred into a polyvinylidene fluoride membrane (GE Healthcare), blocked with 5% skim milk, and incubated with the primary antibodies (1:1000 dilution). Antibodies against caspase-3, -8, -9, Bax, Bcl-2, Bcl-xL, HIAP-1, HIAP-2, p53, p21, E2F1 and p73 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). After incubation with the horseradish peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent enhanced chemiluminescence assay kit (GE Healthcare) according to the manufacturer’s instructions. Bands in the blot were visualized using a LAS3000 luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan) [15 (link)].