Figure 5.
Fractionation of caspases activated by FasC or reaper by DEAE-column chromatography. Schneider’s cell transformants for human FasC (A) or reaper (B) were treated with 0.5 mM CuSO4 for 12 hr. Cytosolic extracts (S100) were prepared from these cells. Samples of extract containing approximately 25 mg of protein from the FasC and reaper transformants were fractionated by DEAE-column chromatography with a linear gradient of NaCl from 0 to 0.5 M. Caspase 1-like (○) and caspase 3-like (•) activities in each fraction were determined by using specific fluorescent substrates. The short-dashed lines indicate the absorbance at 280 nm. The fractions containing the peaks of caspase activity from FasC transformants (fractions 15–21) and from reaper transformants (fractions 8–11 and 21–26) were pooled. These samples were designated as ICE-FasC, ICE-RPR-A, and ICE-RPR-B, respectively, and used for subsequent experiments described in Fig. 6.