Figure 5
Quantitative determination of PARP cleavage and degradation in MCF-7 cells following induction of apoptosis by treatment with doxorubicin. The left panel shows the results of vector transfected (Neo) (top), Bcl-2 (middle) and Bcl-XL (bottom) expressing MCF-7 cells treated for 48 hours with increasing doses of doxorubicin as indicated, and cell lysates immunoblotted for PARP. The migration position of the 89 kDa PARP fragment diagnostic of caspase cleavage is indicated to the left of the blots (arrow). The antibody used for immunoblotting does not recognize cleavage products generated by other proteases activated during apoptosis. Therefore, both PARP cleavage and degradation were combined into a single apoptosis index (right panel, top) determined by measuring the band intensities for the116 kDa full length PARP in untreated cells (green box, 0), and in cells treated with doxorubicin (e.g. blue box) for the dose indicated above the lanes, on a Kodak image station. Actin blots were used to demonstrate equal protein loading in each lane (Figure 6). This index is plotted versus dose of doxorubicin for vector transfected cells (neo), and for cells expressing 3.6 ng Bcl-2 and 0.9 ng Bcl-XL per μg of total cell protein, respectively (right panel, bottom). EC50 values determined from the resulting dose response curves are indicated with coloured upward arrows.