For in vivo ubiquitination assay, HEK-293T cells were co-transfected with indicated expressing plasmids. Cells were grown overnight and were then treated with MG132 (10 μM) for 6 h, followed by immunoprecipitation and western blot analyses.
In vitro ubiquitination assay was performed as essentially described [35 (link)]. Briefly, HEK-293T cells were co-transfected with indicated expressing plasmids. Moreover, 36 h post-transfection, cells were treated with MG132 (10 μM) for 6 h before immunoprecipitation with anti-HA beads. Purified FBXO3 on HA-beads were added to the in vitro ubiquitylation mixture containing 50 mM Tris (pH 7.6), 5 mM Mgcl2, 0.6 mM DTT, 2 mM ATP (FLAAS, Sigma-Aldrich), 1.5 ng/μl E1 (UBE1, 23–021, Merck Millipore, Darmstadt, Germany), 10 ng/μl UbcH5a (23–029, Merck Millipore), 10 ng/μl UbcH7 (23–047, Merck Millipore), 1 mg/ml ubiquitin (662057, Merck Millipore), and 1 μM ubiquitin aldehyde (662056, Merck Millipore). Samples were incubated for 2 h at 30°C and subjected to western blot analysis.
In vitro ubiquitination assay was performed as essentially described [35 (link)]. Briefly, HEK-293T cells were co-transfected with indicated expressing plasmids. Moreover, 36 h post-transfection, cells were treated with MG132 (10 μM) for 6 h before immunoprecipitation with anti-HA beads. Purified FBXO3 on HA-beads were added to the in vitro ubiquitylation mixture containing 50 mM Tris (pH 7.6), 5 mM Mgcl2, 0.6 mM DTT, 2 mM ATP (FLAAS, Sigma-Aldrich), 1.5 ng/μl E1 (UBE1, 23–021, Merck Millipore, Darmstadt, Germany), 10 ng/μl UbcH5a (23–029, Merck Millipore), 10 ng/μl UbcH7 (23–047, Merck Millipore), 1 mg/ml ubiquitin (662057, Merck Millipore), and 1 μM ubiquitin aldehyde (662056, Merck Millipore). Samples were incubated for 2 h at 30°C and subjected to western blot analysis.
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