Fig. 4. In vitro Trim-Away reveals direct substrate ubiquitination.

a Schematic showing an in vitro ubiquitination reaction in which ligase (R-R-PS), substrate (GFP) and anti-GFP antibody are incubated together with E2 enzymes Ube2W and Ube2N/Ube2V2 to promote either mono- or polyubiquitination. b Western blot of experiment described in (a). Top panel is blotted for GFP, middle panel for IgG and lower panel for TRIM21. Depending on the E2s present, monoubiquitinated species or a higher molecular weight smear indicative of polyubiquitin are observed. Representative example from n = 2 independent experiments. c Western blot of experiment similar to (b) but comparing R-R-PS to Ac-R-R-PS. Note that while R-R-PS ubiquitinates itself, antibody heavy chain and substrate, Ac-R-R-PS only ubiquitinates antibody and substrate. Representative example from n = 2 independent experiments. Source data are provided as a Source Data file. See also Supplementary Fig. 4.