R960cus

OTU activity towards cellular conjugated ISG15 was determined using purified OTUs and cell lysates. Cell lysates containing conjugated human ISG15 were obtained by treating Huh7 cells with 1000 IU of IFN-β for two days before harvesting in triton lysis buffer (1% triton X-100, 20 mM Tris-HCl, 2.5 mM MgCl₂). Alternatively, lysates were obtained by transfecting HEK293T cells with plasmids expressing V5-tagged sheep or human ISG15 (Genscript), and human Ube1L, UbecH8, and HERC5 using Trans-IT-LT1 (Mirus). Lysates were clarified by centrifugation (20 min at 13,000 rpm) and incubated for 20 min at room temperature with purified OTU. Samples were mixed with 4x Laemmli buffer and boiled at 95°C for 8 minutes. Proteins were separated on 4–12% Bis-Tris SDS-PAGE gels and transferred to nitrocellulose membranes using a trans-blot system (BioRad). ISG15 was detected using an ISG15 antibody (Proteintech # 15981-1-AP) or a V5 tag antibody (ThermoFisher Scientific #R960CUS). Tubulin was used as loading control marker (T5169, Sigma). Primary antibodies were detected with SuperSignal West Dura Fast Western blot kits (Thermo Fisher). Protein bands were visualized using a ChemiDoc MP system (BioRad).

The following antibodies were used: anti-PrP monoclonal antibodies 3F4^{71 (link)}, anti-HA (mAb, MMS-101R; Covance), mouse monoclonal anti-V5 antibody (mAb, R960CUS; Thermo Fisher Scientific), mouse monoclonal anti-GAPDH (mAb, AM4300; Thermo Fisher Scientific), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Thermo Fisher Scientific), IRDye conjugated secondary antibody (IRDye 800CW donkey anti-mouse; LI-COR Biosciences). All standard chemicals and reagents were ordered from Merck/Sigma-Aldrich if not otherwise indicated. The monoclonal anti-HA-agarose beads were purchased from Merck/Sigma-Aldrich and cOmplete Mini EDTA-free Protease Inhibitor Cocktail from Roche.