Figure 5

Cleavage of PM/Scl-75 by recombinant caspases. (a) ³⁵S-labeled PM/Scl-75 translated in vitro was incubated with 200 nM purified murine recombinant caspase-1, caspase-2, caspase-3, caspase-7, caspase-8 or caspase-11 for 1.5 hours at 37°C. The resulting reaction products were analyzed by 10% SDS-PAGE, followed by autoradiography. In the first lane the mock-incubated protein was loaded. The filled arrowhead marks the full-length proteins and the open arrowhead the cleavage products. Note that in this experiment the pCI-neo-VSV-PM/Scl-75c-α cDNA was used for in vitro transcription and translation of PM/Scl-75. The positions of molecular mass markers are indicated in kDa at the right and the positions of the relevant polypeptides at the left. (b) As a control for caspase activity the recombinant caspases were incubated with 50 μM fluorogenic substrate in 200 μl of CFS buffer. The release of fluorescent 7-amino-4-methylcoumarin was monitored for 1 hour at 37°C at 2-minute intervals in a fluorimeter. Data are expressed as the increase in fluorescence (Delta F) as a function of time.