BubR1 autophosphorylation is essential for accurate chromosome segregation. (A) Immunoblotting of mitotic lysates of T98G cells 48 h after transfection with the indicated siRNAs and siRNA-resistant mCherry-BubR1 expression vectors as indicated. endo., endogenous; TA, T608A; TE, T608E. (B) Time spent in prometaphase and metaphase (or pseudometaphase) of transiently transfected HeLa cells (stably transfected with EYFP-H2B) with BubR1 siRNA and indicated plasmids encoding siRNA-resistant mCherry-tagged wild-type (WT) BubR1 or the BubR1 phosphorylation mutants. Each vertical bar represents a single cell. The transfected cells were identified by mCherry before live-cell imaging. Pseudometaphase is designated as when the majority of chromosomes are aligned with an obvious metaphase plate, but a few chromosomes are at the poles. (C) A summary of median time spent in prometaphase and metaphase (or pseudometaphase) as well as the percentage of cells with polar chromosomes (pseudometaphase) and chromosome missegregation during anaphase onset from data presented in B. (D and E) Stills of live-cell imaging showing a cell in which endogenous BubR1 was replaced with the nonphosphorylatable BubR1T608A mutant (D) or the phosphomimetic BubR1T608E mutant (E) during unperturbed mitoses. Bar, 10 µm.