Atp flaas

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.

For in vivo ubiquitination assay, HEK293T cells were co-transfected with expressing plasmids encoding Flag-EGFR, FBXL2, and either wild-type HA-ubiquitin, HA-ubiquitin-Lys 48-only or HA-ubiquitin-Lys 63-only. Cells were grown overnight and were then treated with 20 μM MG132 for 4 h before harvesting. Cell lysates were immunoprecipitated using anti-Flag resin, followed by Western blot analyses.
In vitro ubiquitination assay was performed as described^{28 (link)}. Briefly, HEK 293T cells were co-transfected with Flag-EGFR and HA-FBXL2 or HA-FBXL2^∆F expressing plasmids. Thirty-six hours post transfection, cells were treated with 20 μM MG132 for 4 h before immunoprecipitation with anti-HA beads, which were then added to the in vitro ubiquitylation mixture containing 0.1 μM E1 (UBE1, 23-021, Merck Millipore), 0.25 μM Ubch3 (23-022, Merck Millipore), 0.25 μM Ubch5c (23-035, Merck Millipore), 2 mM ATP (FLAAS, Sigma-Aldrich) in the presence or absence of 1 μM ubiquitin aldehyde (Millipore, 662056) and 2.5 μg/μL ubiquitin (Millipore, 662057). Samples were incubated for 2 h at 30 °C and analyzed by immunoblotting.