Ubch13 uev1a

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.

In vitro ubiquitination of ERK1 or ERK1^2KR was performed at 37 °C for 60 min in 50 μL reaction buffer (50 mM Tris pH 7.5, 5 mM MgCl₂ and 2 mM DTT) which contains purified Flag-ERK1 or ERK1^2KR protein (5 μM), UBE1 (100 nM, Boston Biochem), UbcH13/Uev1a (1 μΜ, Boston Biochem), His-ubiquitin or His-ubiquitin K63R (100 μΜ, Boston Biochem), Flag-TRIM15 (2 μM) and Mg²⁺-ATP (10 mM, Sigma). The reaction mixtures were heated with addition of SDS-loading buffer at 95 °C for 10 min and then diluted with buffer (0.05% Triton, 0.1 M Na₂HPO₄/NaH₂PO₄, 10 mM imidazole, pH 8.0) for purification of ubiquitinated ERK1 or ERK1^2KR by Ni-NTA beads (Qiagen). Eluted proteins were analyzed by immunoblot.