Expression of a catalytically inactive Smurf2 impairs the spindle assembly checkpoint with Mad2 destabilization. (a) Immunoblotting for mitotic regulators in HeLa cells transfected with Flag-tagged Smurf2(C716A) or a control plasmid. Cells were synchronized at S phase with thymidine treatment, released into a nocodazole-containing medium, and harvested after incubation at the indicated times after release (see Materials and methods). OE, overexpression. (b) Chromosomal misalignment at metaphase and lagging chromosomes at anaphase observed in cells expressing Smurf2(C716A). Immunofluorescence microscopy of cells stained with anti-Flag antibody for exogenously expressed Smurf2, ACA, and DAPI. Cells were harvested 8.5 h after release from the thymidine block. The second row shows misaligned chromosomes at metaphase, and the bottom row shows lagging chromosomes at anaphase in cells expressing Smurf2(C716A). Bars, 10 μm. (c) Percentages of cells with chromosomal defects in control cultures (open bars) and Smurf2(C716A)-transfected cultures (shaded bars). Data are means ± SEM (error bars) from three independent experiments.