Smurf2 is required for stability and functional localization of Mad2. (a) Smurf2 silencing rapidly down-regulates Mad2 protein but not mRNA. HeLa cells in asynchronous culture were transfected with Smurf2 siRNA or nonspecific dsRNAs (siNS) and harvested at the indicated times after transfection for Western blotting and RT-PCR. (b) Smurf2 silencing results in increased polyubiquitination of Mad2 followed by proteasomal degradation. HeLa cells were transfected with a Mad2 expression plasmid and Smurf2 siRNA #1 or control dsRNA. Cells were then treated with 2 μM MG132 for 4 h and analyzed by immunoprecipitation (IP) with Mad2 antibody followed by Western blotting using the indicated antibodies. (c) Smurf2 physically interacts with Mad2. Untransfected HeLa cells were released from synchronization at S phase with double-thymidine treatment and were harvested at the indicated times for immunoprecipitation followed by Western blotting. Ig, Ig heavy chain; NS, nonspecific band. (d) Ectopic expression of Mad2 is unable to restore the spindle assembly checkpoint in Smurf2-depleted cells. Western blotting for mitotic regulators in HeLa cells transfected with the indicated plasmid and siRNA. Cells were synchronized with thymidine treatment, released into a nocodazole-containing medium, and harvested at the indicated times after release (see Fig. 4 b and Materials and methods). (e) Ectopically expressed Mad2 localizes at centromeres in nocodazole-treated control cells but not in Smurf2-depleted cells. Immunofluorescence microscopy with anti-Mad2 and ACAs 10 h after release from the thymidine block. The close-up images are shown with threefold higher magnifications. Bars, 10 μm.