Figure 2. TIF1γ association with the APC/C and Cdc20 is cell-cycle dependent.

(A) TIF1γ binds the APC/C preferentially in mitosis and S phase. HeLa whole cell extracts were prepared from G1, S, G2 and M phase enriched cell populations. APC7 was then immunoprecipitated from each fraction, isolated upon Protein G Sepharose, and the relative amount of TIF1γ associated with APC7 immune complexes was determined by Western blot. Cell cycle status was verified by Western blotting for cyclins A, B, phospho Ser10-Histone H3 (pH3) and α-tubulin. (B) The ability of TIF1γ to bind the APC/C decreases once the SAC is satisfied and cells progress through mitosis. HeLa cells were treated with 200ng/ml nocodazole for 20h. Mitotic cells were then isolated by shake-off and either harvested, or released back into cycle following the removal of nocodazole and then harvested at later times. APC7 was then immunoprecipitated from cell lystaes, isolated upon Protein G Sepharose and TIF1γ binding to the APC/C was assessed by Western blot. Levels of BubR1, cyclin B1, APC4, APC7, TIF1γ and α-tubulin were also assessed by Western blot. (C) TIF1γ binds Cdc20 preferentially in mitosis. HeLa whole cell extracts were prepared from G1, S, G2 and M phase enriched cell populations. Cdc20 and TIF1γ was then immunoprecipitated from each fraction, isolated upon Protein G Sepharose, and the relative amount of TIF1γ associated with Cdc20 was determined by Western blot. (D) Cdc20 knockdown does not affect APC/C association with TIF1γ HeLa cells were treated with non-silencing siRNA, Cdc20, and Cdh1 siRNAs. Forty-Eight h post-knockdown cells were harvested and subject to immunoprecipitation with anti-TIF1γ antibodies Following SDS-PAGE TIF1γ association with the APC/C was assessed by Western blotting.