Ubicrest deubiquitinase enzyme kit

The assay was carried out using the UbiCREST Deubiquitinase Enzyme Kit (Boston Biochem). HEK 293 cells seeded in a 15 cm cell culture dish were transfected at 60% confluence with 15 μg DNA containing STREP-tagged Tat, FLAG-tagged PJA2 and ubiquitin by the Poly Jet transfection reagent. After 30 hours, cells were harvested, lysed in Lysis Buffer and rocked for 30 minutes at 4 °C. Lysate was centrifuged and supernatant was incubated with Streptactin Superflow resin slurry (IBA-GmBH) for 3 hours at 4 °C. The resin was washed sequentially with high salt WASH buffer (Phosphate-Buffered Saline +1 M NaCl +0.05% Tween-20) for 4 times, PBS-T (Phosphate-Buffered Saline +0.05% Tween-20) for 2 times, and DUB reaction buffer (1 ml) for 2 times, which was supplied with the kit, and eventually resuspended in DUB buffer (total volume was 450 μl). Resin was distributed equally among 9 microcentrifuge tubes on ice and 45 μl of DUB reaction buffer was added. The reactions were started by adding 5 μl DUB enzyme or 5 μl DUB buffer (control reaction) separately, followed by mixing and incubation at 37 °C for 30 min. After the reaction, collected resin was mixed with 50 μL of 1× Laemmli sample buffer (containing 2-Mercaptoethanol) and boiled before loading onto SDS-PAGE gel. Ubiquitination of Tat was verified by immunoblotting with anti-Tat antibody.

Prior to the deubiquitinase reactions, immunoprecipitations were performed using similar conditions to the ones described above for ubiquitination-related pull-downs. Briefly, HEK293T TRIM25 CRISPR KO cells were plated in 6 well plates and transfected with 500ng of either pcDNA3.1-YFP or pcDNA3.1-TRIM25^CR in combination with 125ng of pCAGGS-VP35, 90ng of pCAGGS-VP30, 1.2μg of pCAGGS-L and 125ng of pCAGGS-NP, in the absence of ectopically expressed ubiquitin. Cells were lysed 48 hours post-transfection and lysates immunoprecipitated overnight with rabbit anti-EBOV-NP antibody using protein G agarose beads. Following immunoprecipitation, beads were treated with UbiCREST Deubiquitinase Enzyme Kit (BostonBiochem) following manufacturer’s instructions. Briefly, beads were washed several times before being equally split into separate tubes and treated with either USP2 or left untreated (NTC, non-treated control), for 30 minutes at 37°C. Input lysates and pull-down beads were subjected to SDS-PAGE and Western blots performed using antibodies against HSP90 (Santa Cruz), TRIM25 (Abcam) and EBOV-NP. Blots were visualized by ImageQuant using anti-mouse or anti-rabbit HRP-linked antibodies (Cell Signaling).