Ectopic overexpression of Smac/DIABLO does not enhance Apo-2L/TRAIL–induced DISC and its processing of caspase-8.

Jurkat cells were transiently transfected with either the control vector (pcDNA3.1 Zeo) or pcDNA3.1-expressing Smac/DIABLO (pcDNA3.1 Smac). Twenty-four hours after transfection, cells were treated with either 100 ng/mL Apo-2L/TRAIL for 2 hours or 10 nM Epo B for 4 or 24 hours. Following this, cells were harvested and cell lysates were obtained. These were either immunoprecipitated with anti-DR4 and anti-DR5 antibodies and immunoblotted with anticaspase-8 antibody to evaluate its recruitment to and processing by DISC (A), or the cell-lysates were immunoblotted with anticaspase-8 and β-actin antibodies (β-actin serving as the loading control) (B). Immunoblots are representative of results from 3 experiments.