Extended Data Fig. 5: Development of TIRF-based APC/C-dependent substrate ubiquitination assay following fluorescently-labeled UBE2C.
a, b – UMAP representation showing the particle distribution of the sub-states in the ‘CRL Down’ (A) and ‘CRL Up’ (B) major states. c – TIRF microscopy movies capture substrate-E2 interactions prior to complete substrate depletion. Experimental mixtures for the given conditions were split evenly between microscopy flow cells for data capture (Fig. 3d) and a ubiquitination reaction with 50 nM Biotin-CycBN*-A488 spiked in, shown above, that was ran on an SDS-PAGE gel and fluorescently scanned. The banding of the ubiquitinated substrate is distorted due to the high BSA concentration needed for microscopy. The experiments were performed independently three times. d – Duration of detected binding events in seconds across multiple experiments and with increasing concentration of UBE2S CTP. e – There is minimal background signal from slide functionalization (left) and robust signal from immobilized substrate (center left) in the Substrate channel, as well as minimal background fluorescence from the substrate in the UBE2C channel (center right). The addition of UBE2C to immobilized substrate without APC/C-CDH1 shows minimal non-specific UBE2C binding (right). f – Example images showing UBE2C binding events upon addition of APC/C-CDH1 and increasing levels of UBE2S CTP. g – Montage showing representative time series of a UBE2C binding event in the UBE2C channel at 200 ms intervals. h – Montage (top) depicting duration of individual binding events in experiments described in (C) indicated by red or blue asterisk. Trace of raw intensity values (bottom) showing individual UBE2C binding events; red or blue asterisks denote corresponding binding events shown in montage (top).
