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 described28 (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.
In vitro ubiquitination assay was performed as described28 (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.
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