Flag trim15

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.