In Vitro Ubiquitination of GFP-PTOV1

In vitro ubiquitination assays were performed as previously described (40 (link), 41 (link)). In brief, recombinant human HUWE1 HECT domain was produced and purified from BL21 bacterial cells. Recombinant E1, UbcH7 (E2), and ubiquitin proteins were purchased from R&D Systems. GFP-PTOV1 was overexpression in HEK-293T cells by transiently transfecting 16 ug of pcDNA3 encoding GFP-PTOV1 into 1×10⁶ cells in a 10cm dish. 48 hours later, cells were lysed and GFP-PTOV1 was retrieved with GFP-TRAP resin (Chromotek, Planegg, Germany). After washing, GFP-PTOV1 on beads was incubated at 30°C for 3 hours with 10 ng of recombinant E1, 100 ng of recombinant UbcH7, 100 μg of ubiquitin, and 1 μg of a purified HECT domain of HUWE1 in 40 μl of reaction buffer [50 mM tris (pH 7.5), 5 mM MgCl2, 2 mM ATP, 2 mM DTT]. After the incubation, the beads were washed 3X with a buffer containing 10 mM Hepes (pH 7.4), 150 mM KCl, 1% NP-40, and 400 mM NaCl. GFP-PTOV1 protein was eluted with Laemmli SDS sample buffer and subjected to Western blot.