Figure 2. Characterization of the apoptotic machinery in p65 null MEFs.. (A) Expression of several key components of the apoptotic machinery in p65 null and reconstituted cells was compared. Expression of Apaf-1, caspase-2, cytochrome c, and XIAP was detected by immuno-blot. Expression of caspase-3 and -9 was assessed by RT-PCR from total RNA extracted from the cells as indicated. (B) S-100 extracts from p65 null (vector) and reconstituted cells (p65) were incubated with 1 mM ATP and 1 μM equine cytochrome c at 37 °C for 1 hr. Caspase activity was then assessed by cleavage (arbitrary fluorescence units per minute [AFU/min]) of the fluorogenic substrate, Ac-DEVD-afc. (C) caspase-9 processing by autoradiography. S-100 extracts were incubated under the conditions described above with in vitro translated caspase 9 and subjected to SDS-PAGE.