FIG. 2.
Smac deficiency impairs caspase 3 cleavage in vitro. (A) Impaired in vitro caspase 3 cleavage in Smac−/− MEFs. Lysates of Smac+/+ or Smac−/− MEFs were incubated in vitro with the indicated assay reagents, and the cleavage of procaspase 3 was detected by Western blotting with antibodies recognizing either the cleaved (active) form of caspase 3 or procaspase 3. One of the representative results from three independent samples is shown. cyt C, cytochrome c. (B) Restoration of procaspase 3 cleavage by addition of recombinant Smac protein. Lysates of Smac−/− MEFs were incubated with the indicated reagents plus 1 nM to 1 μM recombinant Smac protein. Detection of activated caspase 3 was as for panel A.