Supplementary Figure 3

(A) Calibration of Cdc20 T70 phospho-specific antibody. GST-Cdc20 47–78 wt was phosphorylated in vitro with cdk1-cyclinB using [γ-³²P] labeled ATP. The sample was blotted on PVDF and the Cdc20 T70 signal determined by quantitative western blot and then analyzed by autoradiography. Cdc20 T70p antibody signal was normalized to [³²P] signal and the value obtained from the Cdc20 3S was multiplied by the Cdc20 wt to determine how well the T70p antibody recognizes the S70p epitope. The obtained value was used to calculate to what extent Cdc20 3SP was phosphorylated in cell extracts. Values are quantified signal intensities. The experiment was performed once. (B) The APC/C was purified from nocodazole arrested cells using a monoclonal APC4 antibody and then control treated or treated with lambda phosphatase as indicated. Different amounts of purified APC/C was analyzed by western-blot and probed with the indicated antibodies. Data show 1 out of 2 independent experiments with similar results. (C) As in B with cells depleted of either APC1 or APC3 using specific RNAi oligoes. Note that depletion of APC1 destabilizes the APC/C. Data show 1 out of 2 independent experiments with similar results. (D) HeLa cells synchronized using a double thymidine arrest and release protocol were released from the final thymidine block (t = 0) and the APC/C purified at the indicated times. The samples were analyzed by western-blot with the indicated antibodies. The level of APC1 S355p intensity was normalized to APC4 intensity on the diagram to the right. Data show 1 out of 2 independent experiments with similar results. (E) HeLa cells were transfected with YFP-Cdh1 wt or mutants thereof and then YFP-Cdh1 proteins were isolated from mitotic cells using a YFP affinity resin. YFP-Cdh1 wt sample was treated with lambda phosphatase as indicated and then different amounts of purified YFP-Cdh1 proteins were analyzed by western-blot using the indicated antibodies. The quantification is shown for 8 ul sample as this allows for more accurate quantification. Data show 1 out of 2 independent experiments with similar results.