Extended Data Fig. 7: Ub binding to coactivator serves multiple purposes during APC/C-dependent polyubiquitination.
a – Quantitation of CycBN* modified with >4 Ubs from single encounter assays in Fig. 5c. Error bars: SEM. The experiments were performed independently three times. b – Substrate-independent, APC/C-dependent hydrolysis of an oxyester-linked Ub from UBE2C C114S is not impeded by the addition of UbVCDH1, monitored by Coomassie-stained SDS-PAGE gel. The experiments were performed independently three times. c – Synthesis of di-Ub by APC/C-UBE2S is not inhibited by UbVCDH1 as revealed by scanning of an SDS-PAGE gel where the acceptor Ub* is fluorescently labeled. The experiments were performed independently three times. d – UbVCDH1 inhibits APC/C-CDH1-dependent ubiquitination activity of all substrate and E2 combinations tested, monitored by fluorescent scanning. The experiments were performed independently three times. e – Ubiquitination of Ub-Securin and Ub-Securin DMut as shown in Fig. 5c. The experiments were performed independently three times. f – Similar to Fig. 5f, ubiquitination of *UbVCDH1 requires UBE2C and does not occur with UBE2S alone, as monitored by fluorescent scanning. Uncropped gels representative of n≥3 independent experiments for B-F available in source data.
