E1-E2 Thioester Transfer Assay

E1-E2 thioester transfer assay was adapted from the previously described protocol^{33 (link)}. Assays were performed with 20 nM E1 (species-matched to E2), 500 nM E2, 5 μM Ub, and 1 mM ATP in 20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl₂ buffer. Reaction was started by addition of ATP and mixing then quenched after 30 s by addition of 2x UREA sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) buffer. Owing to inherently low activity, Ubc15 thioester transfer assay was conducted with 50 nM E1 for 60 s. Samples (7.5 μl each) were subjected to SDS-PAGE at 150 V. Gels were then stained with SYPRO Ruby (BioRad) and visualized with a ChemiDoc MP (BioRad). Data quantification was conducted using densitometry in ImageJ software with original unedited images. Densitometry measurements were normalized as a percentage of the control WT assay on the same gel. Data are represented as an average of 3–4 technical replicates with + /−1 standard deviation error bars. Unprocessed images of representative gels for all biochemical assays are provided in the Source Data file.