Cytosolic expression of mature Smac variants and the interaction of Smac56, Smac60, and Smacδ with XIAP following transient expression.

<p>(<b>A</b>) Cells were stably or transiently transfected with IRES2.eGFP expression plasmid (<i>Stable</i> versus <i>Transient</i>) containing Ub-Smac56 or full-length Smac 1-239. 911 cells were subjected to hypotonic lysis, and cytosol and mitochondrial fractions were prepared by differential centrifugation. Proteins were resolved by SDS-PAGE and subjected to western blot analysis with a V5 antibody to detect ectopic Smac. (<b>B</b>) Cells were transiently cotransfected with the indicated GST vector (pGFLEX) and the indicated HA-Ub-Smac (pcDNA3.1-V5His). Two days later cells were lysed, and proteins were purified using GSH-agarose. Lysate proteins (<i>Input</i>) and proteins eluted from the GSH-agarose (<i>Pulldown</i>) were resolved by SDS-PAGE and subjected to western blot analysis with antibody to the V5 epitope or XIAP.