Fig. 6.

Residue Ser92 on Cdc20 is a PP2A^B56 substrate that influences Cdc20 binding to APC/C. (A,B) siCtrl and siB56 cells were collected by mitotic shake-off. Lysates (lanes 1,2), and control IgG (lane 3) or Cdc20 IPs (lanes 4,5) were analyzed by western blotting for the indicated proteins. (C) Quantification of three replicate experiments described in A,B. Data are mean±s.d. n.s., not significant (P>0.05), **P<0.005, Student's two-tailed t-test. (D) Ser92 is a determinant of Cdc20 binding to APC/C. HeLa cells expressing doxycycline-inducible FLAG–Cdc20 wild type (WT), or the mutants S92A or S92E were collected by mitotic shake-off, lysed and analyzed directly (lanes 1-3) or used in a FLAG (lanes 5-7) or control IgG (lane 4) IP and probed for the indicated proteins by western blotting. (E–G) Cdc20^S92A expression does not rescue mitotic exit delays in siB56 cells. (E) Schematic of B56 depletion and FLAG-Cdc20 induction in HeLa cells. (F) Mitotic siCtrl and siB56 cells expressing FLAG–Cdc20 constructs were harvested as outlined in E and analyzed alongside control mitotic HeLa lysates by western blotting. endog, endogenous. (G) Cells were imaged live and duration of mitosis was scored. The cumulative fraction of cells that exited mitosis as a function of time after mitotic entry is plotted. 50 cells were scored per condition. Experiments in D,F,G were performed in triplicate. (H) Schematic of PP2A^B56-dependent stimulation of APC/C association with Cdc20.