2.3. In vitro ubiquitination assay

We set up a ubiquitination assay following previously described methods (Murata et al., 2005; Sun et al., 2012). Briefly, 2 μg of GST‐mot‐2 was incubated in a 100 μL reaction mixture containing 50 ng of (His)₆‐UBE1 activating enzyme (E1; Boston Biochem, Cambridge, MA, USA), 0.5 μg of (His)₆‐UbcH5b Ub‐conjugating enzyme (E2; Enzo Life Sciences, Farmingdale, NY, USA), 2 μg of GST‐CHIP ubiquitin ligase (E3; Ubiquigent, Dundee, Scotland, UK), and 5 μg of GST‐ubiquitin (Boston Biochem). These optimized micrograms of recombinant proteins in the prepared reaction were based on the molecular weights of individual proteins. A reaction buffer (50 mM Tris/HCl, pH 7.5, 4 mM ATP for energy source, and 2 mM MgCl₂) containing recombinant E1, E2, E3, GST‐ubiquitin, and GST‐mot‐2 received different concentrations of recombinant (His)₆‐UBXN2A (0, 0.5, 2, and 4 μg) followed by 30 min incubation in a 37 °C water bath. After the in vitro ubiquitination assay, the ubiquitinated mot‐2 was pulled down by p62‐UBA (Enzo) immobilized on agarose beads (Rezvani et al., 2007, 2009). The UBA domain of p62 is able to bind multi‐ubiquitin chains and pull down ubiquitinated proteins. Yield proteins were subjected to 4–20% gradient SDS/PAGE followed by WB using various antibodies, including an anti‐mot‐2 antibody, to determine the level of polyubiquitinated mot‐2. To exclude the possibility that the GST self‐dimerization may promote artificial ubiquitination of mot‐2 proteins, we used agarose beads containing no immobilized UBA domain as control. The increased high‐molecular ubiquitinated mot‐2 in the presence of UBXN2A and the absence of a typical high‐molecular mot‐2 protein in the control lane excluded the possibility that GST self‐dimerization might have occurred and allowed GST‐ubiquitin to conjugate nonspecifically to its substrates.