Western Blotting of Apoptosis Markers

Western blotting was performed as we have described previously [25 (link), 27 (link)]. Briefly, the protein concentrations of samples were determined using the bicinchoninic acid assay reagent (Pierce Chemical Company, Rockford, IL). Thirty micrograms of each protein sample were subjected to western blot analysis using the following primary antibodies: anti-cleaved caspase-3 at 1:2000 dilution, anti-Bcl-2 at 1:1000 dilution, and anti-β-actin at 1:2000 dilution. All antibodies were purchased from Cell Signaling Technology, Beverly, MA, USA. Images were scanned by an Image Master II scanner (GE Healthcare, Milwaukee, WI, USA) and were analyzed using ImageQuant™ TL software v2003.03 (GE Healthcare, Milwaukee, WI, USA). The band signals of other interesting proteins were normalized to those of the corresponding β-actin and then expressed as fractions of the control sample from the same gels.