Figure 3.

The BubR1 Loop Is Required for SAC Function In Vivo

(A) Domain organization of the BubR1 constructs used. Constructs lacking the short (L) and long loop (LL) versions were created by deleting residues 368–379 or 363–396 of BubR1, respectively.

(B) Representative images of HeLa cells transfected with the indicated GFP-BubR1 constructs in presence or absence of endogenous Bub1, showing that the lack of the loop does not influence kinetochore localization, as expected. Cells were treated as in Figure 1E. The scale bar represents 10 μm.

(C) Quantification of BubR1 kinetochore levels in cells treated as in (B). The graph shows mean intensity from three independent experiments. Error bars represent SEM. Values for BubR1^wt in non-depleted cells are set to 1.

(D) FRAP analysis of BubR1^ΔLL in the absence of endogenous BubR1. Relevant recovery parameters are shown. The graph shows mean and SD. The cartoon depicts the expected mode of kinetochore localization of the construct.

(E) Mean duration of mitosis of Flp-In T-REx stable cell lines expressing the indicated GFP-BubR1 constructs in the absence of endogenous BubR1 and in the presence of 50 nM nocodazole. Cell morphology was used to measure entry into and exit from mitosis by time-lapse microscopy (n > 32 for BubR1^Δ(L)L per cell line per experiment) from two independent experiments. Error bars depict SEM.

(F) Western blot of IPs from mitotic Flp-In T-REx cell lines expressing the indicated GFP-BubR1 constructs showing that the lack of the BubR1 loop results in strongly impaired APC/C binding. Vinculin was used as loading control.

See also Figure S4 and Table S1.