In vitro interaction of caspase-8L with FADD and caspase-8a and the inhibition of the binding of caspase-8a to FADD by caspase-8L.

HS1, caspase-8a, and caspase-8L fused at its N-terminus with the polyhistidine epitope (His) were generated by in vitro translation (denoted His-HS1, His–caspase-8a, and His–caspase-8L, respectively). FADD and caspase-8L (not tagged) were also prepared by in vitro translation (denoted FADD, caspase-8L, respectively). His-tagged proteins were incubated with Ni-NTA Magnetic Agarose Beads overnight. Ni-NTA beads bearing His-tagged proteins were washed and resuspended in protein reaction buffer. Next, 50 μL in vitro–translated FADD or caspase-8L was added to each reaction mixture. After 2 hours' incubation, the beads were collected on a magnetic separator. After washing, the bound proteins were eluted by adding 50 μL SDS-PAGE buffer, analyzed by SDS-PAGE, and detected with anti-FADD monoclonal antibody (MBL) (lanes 1-5) or with anti–caspase-8 polyclonal antibody (lanes 6-8). For the blocking experiment (lane 4), His–caspase-8 bearing Ni-NTA beads were incubated with in vitro–translated FADD in the presence of 50 μL translated caspase-8L. In vitro–translated FADD (lane 1) or caspase-8L (lane 6) were incubated with Ni-NTA beads to exclude nonspecific binding of FADD or caspase-8L to Ni-NTA beads. The bands corresponding to FADD or caspase-8L are indicated by arrows.