Persistent cytosolic expression of Smac56, but not Smac60, ablates immunostaining by a monoclonal antibody similarly to certain missense mutations of the dimer interface.

<p>(<b>A</b>) Cell lines that stably expressed the indicated Ub-Smac-V5 plasmids (IRES2.eGFP) and cells that were transiently transfected with Ub-Smac56-V5 or Ub-Smac60-V5 (IRES2.eGFP) were lysed and proteins were resolved by SDS-PAGE. <i>Mock</i> indicates a cell line transfected with empty IRES2.eGFP vector. Western blot analysis was done firstly with the monoclonal Smac antibody and secondly with antibody to the V5 epitope. (<b>B</b>) Cell lines that stably expressed the indicated missense mutation of Ub-Smac56-V5 (IRES2.eGFP) and cells that were transiently transfected with Ub-Smac56-V5 (IRES2.eGFP) were lysed, and proteins were resolved by SDS-PAGE. Western blot analysis was done as indicated for (A).