Monomerization of Cytosolic Mature Smac Attenuates Interaction with IAPs and Potentiation of Caspase Activation
Figure 5
Persistent cytosolic expression of Smac56, but not Smac60, ablates dimerization and interaction with XIAP.
(A) Cells that stably expressed the indicated Ub-Smac-V5 plasmid (IRES2.eGFP) were transiently transfected with the Ub-Smac-GST plasmids (pGFLEX), lysed, and proteins were purified using GSH-agarose. Lysate proteins (Input) and proteins eluted from the GSH-agarose (Pulldown) were resolved by SDS-PAGE and subjected to western blot analysis with antibody to GST-pi and the V5 epitope. (B) Cells that stably expressed the indicated Ub-Smac-V5 plasmid (IRES2.eGFP) were transiently transfected with GST-XIAP plasmid (pGFLEX), lysed, and Input and Pulldown proteins were resolved by SDS-PAGE and subjected to western blot analysis with antibody to XIAP and the V5 epitope as described for (A).