Figure 6.

Bub3 Is Required for Robust SAC Signaling

(A) Domain organization of the BubR1 constructs used.

(B) 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 > 55 per cell line per experiment) from two independent experiments. Error bars depict SEM.

(C) Western blot of IPs from mitotic Flp-In T-REx cell lines expressing the indicated GFP-BubR1 constructs showing a defect especially in binding to APC/C subunits if BubR1 cannot bind to Bub3. Tubulin was used as loading control. ^∗, band resulting from previous incubation with Bub1 antibody; ^∗∗, unspecific band recognized by the Bub1 antibody.

(D) Quantification of the western blot in Figure 6C. The amounts of co-precipitating proteins were normalized to the amount of GFP-BubR1 bait present in the IPs. Values for GFP-BubR1^wt are set to 1. The graph shows mean intensity of two independent experiments. Error bars represent SEM.

See also Figure S4.