Supplemental Material

This article contains the following supporting material:

• Figure 1 - Concentrations of rCaspase-2 added to extracts are comparable to those of endogenous, full-length Caspase-2 in extracts, and rCaspase-2 activity is blocked by zVAD-fmk.
  (A) Jurkat cytosolic extracts (100 μg) and rCaspase-2 were subjected to SDS-PAGE and western blotting using an anti-Caspase-2 antibody. (B) rCaspase-2 (C2) was incubated with or without zVAD-fmk and subsequently tested for its ability to cleave VDVAD-AFC in vitro. Relative fluorescence units versus time are shown. (C) Full length Caspase-2L was in vitro-transcribed and translated from pTARGET-CASP2L (Droin et al., 2001) and labeled with ³⁵S-methionine. 5000 counts were then subjected to rCaspase-2 cleavage for 4 h. (D) Wild-type MEF cytosolic extracts (10 mg/mL) and mouse liver mitochondria (80 μg) were incubated with the indicated recombinant caspases with or without zVAD-fmk for 2 h at 37° C. Supernatants and mitochondrial pellets were resolved by SDS-PAGE and immunoblots were probed with an anti-cytochrome c antibody (Pharmingen).

• Figure 2 - rCaspase-2 induces DEVDase activity in cytosolic extracts incubated with mitochondria.
  rCaspase-2 was incubated with Xenopus laevis egg cytosolic extracts (20 mg/mL) alone, Xenopus laevis egg mitochondria (4%) in Buffer A or with both for 2 h. Samples were subsequently assayed for DEVD-AFC cleavage. Relative fluorescence units versus time are shown.

• Figure 3 - Caspase cleavage of recombinant Bid.
  2 μg of recombinant GST, GST-WT Bid, and GST-D59E Bid were incubated in Buffer A with the indicated recombinant caspases for 3 h at 37° C. Samples were resolved by SDS-PAGE and visualized by Coomassie Blue. (We note that, in contrast to WT Bid (Fig. 4), GST-Bid was cleaved with nearly the same efficiency by Caspases-2 and –8; the bulky GST domain thus appears to alter the presentation of the cleavage site. Therefore, this result using an artificial fusion protein likely does not reflect the properties of native Bid.)