Fig. 5.

B56α interacts with the APC/C constitutively and independently of BubR1. (A,B) B56α co-precipitates with APC/C. (A) Mitotic (mit.) or asynchronous (asy.) HeLa cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting for the indicated proteins. (B) Quantification of three replicate experiments as described in A. Values were normalized to those for the PP2A catalytic subunit (PP2A cat.). (C,D) Mitotic interaction of B56α and APC/C does not require the KARD of BubR1. BubR1 depletion and rescue was performed as described in Fig. 4C, and cells were subjected to IP for B56α (lanes 4,5) or with control IgG (lane 3) and probed by western blotting. (D) Quantification of three replicate experiments as described in C. (E,F) An LxxIxE motif mediates association of APC/C with B56α. Expression of YFP–B56α WT or an R222E mutant was induced by doxycycline in HeLa FLP-In lines, and mitotic cells were collected. (E) IPs for GFP (for YFP-tagged constructs; lanes 4,5) and control (lane 3) IPs were analyzed by western blotting. Asterisk indicates non-specific band. (F) Quantification of three replicate experiments described in E. Data are mean±s.d. n.s., not significant (P>0.05); *P<0.05, **P< 0.005, ***P<0.0005, ****P<0.00005, Student's two-tailed t-test.