Extended Data Fig. 5: UBE2SCTP stimulates the APC/C to activate UBE2C~Ub hydrolysis.
From: Ubiquitin chain-elongating enzyme UBE2S activates the RING E3 ligase APC/C for substrate priming
a, UBE2SCTP promotes a substrate-independent APC/C activation of UBE2C, monitored by the hydrolysis of UBE2S~Ub over time into UBE2C and Ub. Detected using Coomassie-stained gels, full gel from (3d). b, Western blot of (3e) showing successful knockdown of UBE2S and CDC20 in HeLa H2B-GFP. c, Time necessary for the metaphase to anaphase transition for different U2OS cells. Data were collected in 2 independent experiments, total number of analyzed cells n = 67 (control), n = 76 (UBE2S knockdown), n = 67 (CDC20 knockdown) and n = 62 (UBE2S and CDC20 knockdown). Median, 25% and 75% percentiles are shown. ** indicates p<0.001, **** indicates p<0.0001 as calculated with Kruskal Wallis test followed by Dunn’s multiple comparison test. Right panel, western blot showing successful knockdown of UBE2S and CDC20 in U2OS H2B-mCherry. d, Western blot in (3g) with the addition of levels of endogenous or recombinant variants of UBE2S. Uncropped images for panels (a,d) and data for the graph in (c) are available as source data.
