Author: Swatek, Kirby N.; Aumayr, Martina; Pruneda, Jonathan N.; Visser, Linda J.; Berryman, Stephen; Kueck, Anja F.; Geurink, Paul P.; Ovaa, Huib; van Kuppeveld, Frank J. M.; Tuthill, Tobias J.; Skern, Tim; Komander, David
Title: Irreversible inactivation of ISG15 by a viral leader protease enables alternative infection detection strategies Document date: 2018_3_6
ID: 3s86w4iw_22
Snippet: Cloning and Protein Purification. ISG15 and Met1 diubiquitin were cloned into an His-tagged expression vector (32 The catalytic activity of Lb pro was also monitored using an antibody that recognizes GlyGlymodified Lys residues. Loading controls are in Fig. S6C. (B) Infection assays using an FMDV Lb pro -mengovirus chimera. The catalytic activity of Lb pro was monitored as in A, with the difference being that ISG15 was detected using an anti-ISG1.....
Document: Cloning and Protein Purification. ISG15 and Met1 diubiquitin were cloned into an His-tagged expression vector (32 The catalytic activity of Lb pro was also monitored using an antibody that recognizes GlyGlymodified Lys residues. Loading controls are in Fig. S6C. (B) Infection assays using an FMDV Lb pro -mengovirus chimera. The catalytic activity of Lb pro was monitored as in A, with the difference being that ISG15 was detected using an anti-ISG15 antibody (Left). A catalytically inactive FMDV Lb pro -mengovirus (C51A) was used as a control. Fig. S6D shows loading controls. (C) FMDV infection time course using BHK fibroblast cells transfected with FLAG-ISG15 and the ISG15 conjugation machinery. Lb pro catalytic activity was monitored with anti-FLAG (Top) and anti-GlyGly (Bottom) antibodies. All assays were performed in triplicate.
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