2.8. In vitro ubiquitin conjugation assay

Fifty-microgram SH-SY5Y cell lysates were incubated with 1 mM HA-tagged Ub for 1 h in reaction buffer containing 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 5 mM MgCl₂, 10% glycerol, 1 mM DTT, 50 mM MG-132, and 5 mM ub-aldehyde (Boston Biochemicals, Ashland, USA). Ub conjugates were immunoblotted by HA antibody.