High-resolution, high-sensitivity detection of active caspases in apoptotic HL-60 cell extracts by two-dimensional gel electrophoresis. (A) Mobilities of Z-EK(bio)D-aomk reactive caspases on two-dimensional PAGE. Caspases were partly purified from extracts of apoptotic HL-60 cells (see Materials and Methods), labeled with Z-EK(bio)D-aomk, resolved by 2-dimensional PAGE using an immobilized pH gradient, transferred to nitrocellulose membrane, reacted with peroxidase-conjugated streptavidin, and detected by ECL. (B) Indexing of active caspases purified from HL-60 cytosol. Shaded circles correspond to species previously shown to comigrate with known caspases. Spots A1, C1, C3 comigrate with Caspase-3, while spot B1 comigrates with Caspase 6.48 The migration of relevant molecular-weight markers is shown at the left of (A) and (B). (C) Isoelectric points of the detected caspases. (D) Control experiments performed with ortho[³²P]phosphate labeled extracts showed the presence of Z-EK(bio)D-aomk reactive species in extracts prepared from etoposide treated cells (lanes 2, 2′), but not in extracts from control healthy cells (lanes 1, 1′).