Ubiquitination Assays for SOD1 Mutants

In vitro ubiquitination assays were performed as previously described [35 (link)]. Briefly, purified His-tagged SOD1 WT or mutants and GST-tagged parkin or GST were incubated in reaction buffer (50 mM Tris–HCl pH 7.6, 5 mM MgCl₂, 100 mM NaCl, 25 μM ZnCl₂, 2 mM dithiothreitol, and 4 mM ATP) containing E1 enzyme (Boston Biochem), E2 enzymes (UbcH7, UbcH8, or UbcH13/Uev1a, Boston Biochem), and ubiquitin WT or ubiquitin mutants (Boston Biochem). After incubating for 3 h at 37 °C, the reaction was stopped by adding loading buffer, and ubiquitinated SOD1 was detected by immunoblotting with anti-ubiquitin and anti-SOD1 antibodies. In vivo ubiquitination assays were performed as described [38 (link), 49 (link)] in SH-SY5Y cells expressing indicated epitope-tagged SOD1 WT or mutants, parkin, HA vector, and HA-tagged Ub WT or mutants. SOD1 WT or mutant proteins were isolated by immunoprecipitation under denaturing conditions, and their ubiquitination status was assessed by immunoblotting with anti-HA antibody.