Cdc20 T70 is dephosphorylated by BubR1 bound PP2A-B56. (A) Schematic indicating Cdc20 and PP2A-B56 binding to BubR1. The mutations in BubR1 2A are indicated. (B) Stable cell lines expressing Venus-BubR1 WT or 2A were depleted of BubR1 by RNAi and arrested in mitosis with nocodazole. Cdc20 was immunopurified from the cells and the degree of Cdc20 T70p and APC3 T447p determined. Samples were compared with similar samples from the parental cells not treated with BubR1 RNAi. (C) Cdc20, BubR1, and APC4 were immunopurified from nocodazole arrested cells and the level of Cdc20 T70p and APC3 T447p determined. The signal from the phosphospecific antibody was normalized to the pan-specific antibody for Cdc20, and for APC3 T447p the signal was normalized to APC4. For Cdc20, two different antibodies were used. For all experiments, at least three independent experiments were conducted and quantified. Ctrl, control.