Figure 4.

Spindly motif mutants are retained together with Mad1 and Mad2 on kinetochores of aligned chromosomes. (A) Images from a time-lapse imaging sequence of cells expressing histone H2b:mRFP and either GFP:^RRSpindly^WT or GFP:^RRSpindly^F258A. Cells were treated with siRNAs for 32 h, and expression of the Spindly transgenes was induced for 16 h before filming. Bar, 5 μm. (B,C) Immunofluorescence images of cells with congressed chromosomes expressing GFP:^RRSpindly^WT or GFP:^RRSpindly^F258A stained for GFP and Mad1 (B) or BubR1 (C). Bars, 5 μm. (D) Quantitation of checkpoint protein levels at kinetochores of aligned chromosomes relative to unaligned kinetochores in early prometaphase control cells for the indicated cell lines, siRNA treatments, and transgenes.