Figure 3. Non-cleavable caspase-8 is activated most efficiently by FLIP_L and closely approximates endogenous procaspase-8.

(A) Caspase-8 cleaved monomer, cleaved dimer or non-cleavable monomer at 75 nM was incubated with an equimolar amount of FLIP_L in the presence of 20 mM Tris, pH 7.4, 100 mM NaCl (low-salt buffer) or kosmotrope buffer (containing 0.7 M sodium citrate). After 20 min at 37 °C, Ac-IETD-AFC substrate was added at 100 μM and substrate hydrolysis monitored at 37 °C. Results are expressed as fold increase over caspase alone under the respective buffer conditions. (B, C) Endogenous procaspase-8 from a Jurkat cytosolic extract or non-cleavable caspase-8 prepared in hypotonic buffer at a comparable concentration (15 nM) was immunoprecipitated using polyclonal antisera raised against recombinant caspase-8. (B) Immunoprecipitates were assayed for their ability to hydrolyse Ac-IETD-AFC substrate in the presence or absence of 1 μM FLIP_L in either low-salt buffer or kosmotrope buffer as indicated. (C) Western blot of immunoprecipitates using monoclonal anti-caspase-8 antiserum raised against an epitope of the large subunit. (D) Non-cleavable caspase-8, -9 or -10 (500 nM) was added to buffer control, 1 μM of their respective C285A mutant or 1 μM FLIP_L in either low-salt buffer or kosmotrope buffer for 20 min at 37 °C before the addition of substrate (Ac-IETD-AFC for caspase-8, Ac-LEHD-AFC for caspase-9, and Ac-DEVD-AFC for caspase-10).