Fig. 3: Cyclin B1’s phosphate-binding pocket is important for efficient APC/C activation.

A Heat-map visualizing the hierarchical clustering of the 14 significantly enriched proteins identified by quantitative DIA mass spectrometry in the Flag-cyclin B1^wt over Flag-cyclin B1^mut IP after ANOVA (s0 = 0.25, FDR = 0.05) and post-hoc Tukey HSD (FDR = 0.05). Technical replicates of n = 3 biological replicates are averaged and log2 abundances are z-score normalized. Euclidean distances are not shown, for details see methods. B Experimental outline of cell synchronization procedure. Immunoblot assessing α-mNG IP samples of mitotically arrested siB1&B2 HeLa cells expressing mNG-cyclin B1^wt/mut. p150 served as IP control. Right panels show the quantification of the APC3 signal intensity in the input and co-IP samples from n = 3 independent repetitions and plotted against the distance on the blot. C Schematic of experimental design of α-Flag-APC4 IP from mitotic siB1&B2 HeLa cells expressing mNG-cyclin B1^wt/mut and treated for 30 min with 1 µM reversine for SAC silencing. Immunoblot assessing α-APC4 IP samples. D Fluorescent readout (488 nm) of in vitro ubiquitylation assay using immunoprecipitated APC/C from (C), recombinant ubiquitin, UBA1, UBE2C, where indicated UBE2S and as substrate fluorescein-labeled cyclin B1^1–70 (cyc B1^1–70). E Schematic of in vitro ubiquitylation assay using APC/C immunoprecipitated from interphasic Xenopus egg extract. To prevent cyclin B resynthesis following its calcium-induced degradation, extract was co-treated with the translation inhibitor cycloheximide (CHX). Phosphorylation by recombinant PLK1 and CDK1/CKS1/cyclin B1^wt/mut. In vitro kinase reactions were performed in the presence of DMSO or the CDK and PLK1 inhibitors flavopiridol (FL) and BI2536 (BI), respectively. Immunoblot assessing samples taken at indicated steps shown in experimental outline. F Fluorescent readout (488 nm) of in vitro ubiquitylation assay using APC/C purified according to (E) and supplemented with recombinant UBA1, UBE2C, ubiquitin and as substrate fluorescein-labeled cyclin B1^1–70. Immunoblot assessing phosphorylation state of APC3 under different experimental conditions.