Figure 1 - In vitro activation of procaspase-3 by PAC-1.

(a) Progress curves showing the effect of PAC-1 on procaspase-3. 45 l of procaspase-3 (1 M) in 50 mM Tris, pH 8.0, 300 mM NaCl, 500 mM imidazole was incubated with 100 M PAC-1 or vehicle in a 384-well plate at 37 °C. After 2 h, 5 l of Ac-DEVD-pNA in 50 mM HEPES pH 7.4, 100 mM NaCl, 0.10% CHAPS, 10 mM DTT, 0.1 mM EDTA, 10% glycerol was added to each well and the absorbance monitored at 405 nm every 30 s. The final concentration of Ac-DEVD-pNA is 200 m. Data shown are raw data from an experiment performed in triplicate. (b) PAC-1–promoted autoprocessing of procaspase-3 as monitored by western blotting. A solution of procaspase-3 (30 M, with a C-terminal His6 tag) in 50 mM Tris, pH 8.0, 300 mM NaCl, 500 mM imidazole was incubated in the presence of 50 M PAC-1 or vehicle at 37 °C. After 12 h, 2 SDS sample buffer was added (50 mM Tris, pH 6.8, 100 mM DTT, 2% (w/v) SDS, 0.1% bromophenol blue, 10% (v/v) glycerol) and the samples heated to 95 °C. Bands were detected via western blotting for the His6 epitope.