Dithiothreitol (dtt)

GST pull-down assays were performed by mixing GST-Ark2C proteins immobilised on Glutathione Sepharose^TM resin (GE Healthcare) with ubiquitin. Samples were made up to 200 μL in a buffer containing PBS pH 7.4, 0.2% Tween^® 20 (P7949, Sigma-Aldrich), and 2 mM DTT (1114GR005, BioFroxx), and incubated on a tube rotator at 4 °C for 30 min. Samples were then washed four times with PBS before being mixed with reducing 2× SDS sample buffer and resolved by SDS-PAGE. The pulldowns containing Cy3-labelled ubiquitin were imaged using an Odyssey^® Fc (LI-COR Biosciences) with a 600 nm filter prior to Coomassie staining.
Analytical SEC was performed using a 10/300 Superdex 75 increase (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 7.5, 200 mM NaCl buffer. For each run, 100 µL of 200 µM of purified Ark2C variant, ubiquitin, or a mixture of the two proteins was resolved. For mixtures, samples were incubated overnight prior to separation. The recovered fractions were analysed by SDS-PAGE.

Multiturnover activity assays were carried out at 37 °C for the indicated times in 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl₂, 2 mM DTT (1114GR005, BioFroxx), and 10 mM ATP (A7699, Sigma-Aldrich), containing E1 (0.1 μM), E2 (10 μM), E3 (5 μM), and ubiquitin (50–100 μM) (final concentration). The reactions were incubated at 37 °C and stopped by the addition of a reducing SDS sample buffer. For some assays, Cy3-labelled ubiquitin was used in 1:1 ratio with the unlabelled form^{9 (link)}. Following separation by SDS-PAGE reactions containing Cy3-labelled ubiquitin were imaged with an Odyssey^® Fc (LI-COR Biosciences) using a 600 nm filter prior to Coomassie^® Brilliant Blue R-250 (APA1092.0025, AppliChem) staining.
For single-turnover ubiquitin discharge assays, 15 μM of E2~Ub conjugate variants were incubated with 0.125–5 mM l-lysine (L5501, Sigma-Aldrich) and 0.25–1 μM of E3 ligase. All assays were incubated at 25 °C and then stopped by mixing with non-reducing 2× SDS sample buffer, which ensured that the unhydrolyzed thioester E2~Ub conjugate remained intact. Samples were resolved by SDS-PAGE and the reactions containing Cy3-labelled E2~Ub conjugate were imaged by an Odyssey^® Fc (LI-COR Biosciences) using 600 nm filter prior to Coomassie^® Brilliant Blue R-250 (APA1092.0025, AppliChem) staining.