In vitro Rtn4 ubiquitination assay

In vitro Rtn4 ubiquitination assays were described previously.^{15 (link)} For linking recombinant SdeC derivatives to agarose beads, Ni–NTA agarose resin (Thermo Fisher) was washed 3× with 1× ART buffer (20 mM Tris, 10 mM NaCl, pH 7.4) and mixed with His₆-SdeC followed by incubation for 1 h at 4 °C. Next, the SdeC-bound agarose resin was washed 3× with 1× ART buffer before processing to the two-step bead assay. During preincubation, SdeC-bound agarose beads were added to 10 mM Ub, and 100 mM nicotinamide 1, N6-ethenoadenine dinucleotide (ε-NAD, Sigma) in 1× ART buffer (SdeC final concentration 50 nM), and incubated for 1 h at 37 °C. After incubation, the beads were removed from reaction by spin filter columns. The supernatant containing modified Ub and excess ε-NAD were mixed with 400 nM GST-HA-Rtn4 and fresh unbound SdeC variants, then incubated for another hour at 37 °C. Reactions were terminated by addition of reducing loading buffer and boiling.