Fig. 5. p31comet and apcin cooperate to reactivate APC/C.
a, HCT116 cells were treated with 5 nM control or p31comet siRNA, followed by combinations of nocodazole, apcin (0, 6.2, 12.5, 25, 50 μM) or AZ3146 (0, 0.25, 0.5, 1, 2 μM) for 24 hours. The fraction of mitotic cells was determined using a high-throughput fixed cell assay. The mean of four biological replicates from two independent experiments is shown; error bars represent standard deviation. Statistical significance was calculated using a two-tailed unpaired t test (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001). Exact p-values are shown in Supplementary Table 2. b, Reactions were carried out in the presence of APC/C, MCC containing 3Flag-Cdc20-M, 3Myc-His-Cdc20-A, Ube2S, Ube2C, E1, WT ubiquitin, ATP and/or apcin (500 μM) and/or p31comet (1 μM). All components (- substrate) were pre-incubated for 5 minutes followed by addition of fluorescently labeled cyclin A (CycA*). Ubiquitination was analyzed by fluorescence imaging. The experiment was repeated independently with similar results. c, APC/C carrying a Strep tag on APC4 was incubated with strep-tactin resin, 3Myc-His-Cdc20-A, MCC containing 3Flag-Cdc20-M, p31comet (1 μM), apcin (500 μM) or apcin-M (500 μM) for 1 hour and eluates were analyzed by immunoblotting. d, Quantification of experiment in c. Protein abundance was normalized to Cdc27. The fraction of bound protein was set to 1 for the minus compound condition. The mean of five independent experiments is shown; error bars represent SEM. Statistical significance was calculated using a two-tailed unpaired t test (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001). e, MCC containing 3Flag-Cdc20-M was incubated with anti-FLAG-beads, 3Myc-His-Cdc20-A, p31comet (1 μM), apcin (500 μM) or apcin-M (500 μM) for 1 hour and eluates were analyzed by immunoblotting. f. Quantification of experiment in e. The mean of 5 independent experiments is shown. Quantification and representation is identical to d, except protein abundance was normalized to 3FLAG-Cdc20-M. Uncropped images for b, c, e are shown in Supplementary Fig. 8. Exact p-values for d,f are shown in Supplementary Table 2.