Fig. 4: Mapping the SCF^Skp2 interaction site within FLIP(L).

a Co-IP analysis of the interaction of Cullin-1 with full-length FLIP(L), p43-FLIP(L) (p43), FLIP(S), procaspase-8 (Casp8) and FADD; interactions with endogenous Skp1, Skp2 and TRAF2 were also assessed. b Schematic diagram of FLIP expression constructs; the death effector domains (DEDs) and large (p20) and small (p12) subunits of the pseudo-caspase domain are highlighted. The red box indicates the region important for SCF^Skp2 interaction, as deduced from Co-IP experiment mapping the interaction site between FLIP(L) and Cullin-1 and (c) Skp2. d Biotinylated peptide of FLIP(L)’s SCF^Skp2 interaction site pull-down experiment demonstrating the interaction of this region with the SCF^Skp2 complex. e Space-filling and ribbon models of the Cullin-1 interaction site in the large p20-subunit of FLIP(L) and its spatial orientation with respect to the p12 small subunit. f Ribbon models of the Cullin-1 interaction site in FLIP(L) and the corresponding region of caspase-8. g Model derived from the crystal structure (PDB ID: 3H11) of the large and small catalytic domains of the caspase-8/FLIP(L) heterodimer. The position of the SCF^Skp2 complex interaction site and the equivalent (non-contiguous) residues in caspase-8 are highlighted in red. A frontal view of the heterodimer interface is depicted in the middle; the “windows” show 90° rotations of the crystal structure, highlighting the SCF^Skp2 interaction of FLIP(L) and its equivalent region in Procaspase-8.