Ae066

The reaction mixture contains 60 mM Tris (pH 8.5), 50 mM sodium chloride, 50 mM potassium chloride, 10 mM magnesium chloride, 0.1 mM DTT, 3 mM ATP, 90 nM Uba1, 60 μM ubiquitin, and 10 μM (for experiments presented in Figure 3B and Figure 3—figure supplement 1B, C) or 5 μM KlRad6 (for experiments presented in Figure 3C and Figure 3—figure supplement 1A). When indicated, 20 μM KlBre1 RBD (1-206) was supplemented. After incubating at 30°C for the indicated period, the reactions were stopped by boiling in the SDS–PAGE loading buffer. To remove KlBre1 RBD, the boiled reaction mixture was subject to strep-tactin beads precipitation twice and the supernatant was collected for analysis. For comparison, reactions without KlBre1 RBD were supplemented with the same amount of KlBre1 RBD after the reaction and subjected to the same treatment with strep-tactin beads. The reaction mixtures were analyzed with SDS–PAGE and western blot with an anti-ubiquitin antibody (sc-8017, Santa Cruz Biotechnology, RRID: AB_628423, 1:2000 diluted). To probe ubiquitin chains attached to the KlBre1 RBD during the reaction, reaction mixtures prior to the treatment with strep-tactin beads were analyzed with western blot with an anti-strep antibody (AE066, ABclonal, RRID: AB_2863792, 1:5000 diluted).