Evaluation of the reproducibility of caspase quantitation by immunoblotting.

Duplicate blots containing AML samples and a serial dilution of HL-60 cells were prepared as described in the legend to Figure 2. (A) Results obtained when one pair of blots was probed with affinity-purified polyclonal rabbit anticaspase-3 antiserum (top) or monoclonal anticaspase-3 antibody (middle). Histone H1 (bottom) served as a loading control. Responses to therapy are indicated as described in the legend to Figure 2. (B) The blots shown in panel A, along with an additional pair of blots, were probed with both caspase-3 reagents, scanned, and quantitated as described in the “Materials and methods.” A value of 1.0 indicates that a particular leukemia sample contained as much procaspase-3 as an equal number of HL-60 cells after correction for loading by normalization of histone H1 contents.