FIGURE 2.
F1LΔTM inhibits apoptosome-mediated caspase-9 activation and apoptosome assembly. A, procaspase-9 was preincubated with F1LΔTM (lacking GST tag), N1L, XIAP, or Bcl-XLΔTM (1 μm) proteins for 10 min at 37 °C followed by the addition of cytochrome c (Cyt. c), dATP, and recombinant full-length Apaf-1 (1 μm) for 10 min at 37 °C. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). B, procaspase-9 incubated with (+) or without (−) F1L (lacking GST tag) was then added to reactions containing Apaf-1 together with cytochrome c and dATP for 10 min. The samples were size-fractionated by S-200 gel-filtration chromatography, and the resulting fractions were analyzed by SDS-PAGE/immunoblotting (WB) using caspase-9, Apaf-1, or F1L antibodies. The fifth panel represents a chromatography of F1LΔTM alone, which migrated as an apparent dimer. Molecular mass standards are indicated in kDa. C, recombinant F1LΔTM, N1L, XIAP, Bid, Bcl-XLΔTM, or SMAC (10 μm) proteins were preincubated with Apaf-1ΔWD-40 recombinant protein (1 μm) for 10 min, and then procaspase-9 (100 nm) was introduced for 10 min. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). D, various concentrations of F1L (μm) proteins (without GST tag) were preincubated with active caspase-9 (200 nm) preactivated by Apaf-1ΔWD-40 recombinant protein (1 μm) for 10 min. Caspase-9 activity was measured by hydrolysis of Ac-LEHD-AFC (mean ± S.D.; n = 3). RFU, relative fluorescence units.