Detection of ³²P-labeled active caspases in extracts from apoptotic HL-60 cells. (A) Experimental protocol. HL-60 cells labeled with ortho[³²P]phosphate were exposed to etoposide to induce apoptosis. Concentrated extracts from these cells were reacted with Z-EK(bio)D-aomk (±pretreatment with DEVD-fmk) and bound to monomeric avidin-agarose. The input, flow-through, and eluted fraction (with 0.1% SDS) were resolved on a 16% SDS-polyacrylamide gel and then analyzed (B) by autoradiography of the ³²P (lanes 1 through 6) or by ECL (lanes 1′ through 6′). Four percent of the total extract was loaded for lanes 1, 2, 3, 4, 1′, 2′, 3′, 4′. Eighty percent of the purified protein fraction was loaded for lanes 5, 6, 5′, 6′. Lanes 2, 4, 6, 2′, 4′, 6′ correspond to cytosol preincubated with DEVD-fmk before Z-EK(bio)D-aomk labeling. The rectangular areas, A5, A5′, A6 were subsequently subjected to quantitative analysis using the phosphorimager. (C) Quantitation of the phosphorimager data (counts per minute) of areas A5, A6, and densitometry (arbitrary units) for area A5′. The box designated “MERGED” corresponds to a superimposition of A5 and A6, showing the relative position of the ECL bands in A5′ as black rectangles on the x-axis.