FIG. 8.

Cif activity is selectively sensitive to Bid-specific antibodies. (A) A 20-μl volume of S-100 extract from STS-treated HL-60 cells was left untreated (Control) or incubated with 3 μl of rabbit polyclonal antibodies to human Mcl-1 (Mcl-1 AB), Bax (Bax AB), or Bid (Bid AB) at 20°C for 15 min. The extracts were then incubated with HL-60 mitochondria for 15 min at 37°C. The samples were then centrifuged to pellet the mitochondria. The pellet (P) and supernatant (S) were separated, and the cytochrome c content in each fraction was determined by Western blot analysis. (B) Aliquots (100 μg) of S-100 extract prepared from Bcl-2:HL-60 cells were treated with various amounts of caspase 3 or caspase 8 for 60 min at 37°C. The samples were then subjected to electrophoresis and Western blot analyses to determine the status of Bid. (C) Aliquots (100 μg) of S-100 extract prepared from Bcl-2:HL-60 cells were treated with 100 ng of caspase 3 or 25 ng of caspase 8 for 60 min at 37°C. Then the samples were either left untreated (Control) or incubated with 3 μl of rabbit polyclonal anti-human Bax (Bax AB) or Bid (Bid AB) antibodies at 20°C for 15 min. The extracts were incubated with HL-60 mitochondria for 15 min at 37°C, and the samples were centrifuged to pellet mitochondria. Aliquots of the supernatant were subjected to electrophoresis and Western blot analyses for the presence of effluxed cytochrome c (Cyt. c) by caspase 3-activated samples (top) and caspase 8-activated samples (bottom).