Linear linked di ub

5-Amino-2-methyl-N-[(R)-1-(1-naphthyl)ethyl]benzamide (GRL0617) was purchased from Raystar, CN; 5-(acetylamino)-2-methyl-N-[(1R)-1-(1-napthanlenyl)ethyl]-benzamide (compound 6) was purchased from MedChem Express; Z-RLRGG-7-amino-4-methyl-courmarin (peptide-AMC) was purchased from Bachem; Ubiquitin–7-amino-4-methylcourmarin (Ub-AMC) was purchased from Boston Biochem; human ISG15-7-amino-4-methylcourmarin (ISG15-AMC) was purchased from Boston Biochem; Lys6, Lys11, Lys29, Lys33, Lys48, Lys63, and linear linked di-Ub were obtained from Boston Biochem; DL-dithiothroitol (DTT) was purchased from GoldBio; Isopropyl β-D-thiogalactopyranoside (IPTG) was purchased from GoldBio; 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Fisher BioReagents; Imidazole was purchased from Acros Organics; tris(hydroxymethyl)aminomethane (Tris) was purchased from Fisher Scientific; Sodium chloride (NaCl) was purchased from Fisher Chemical; Bovine serum albumin (BSA) was purchased from Sigma Life Science; Dehydrated Luria-Bertani (LB) broth was purchased from Fisher Scientific; Ampicillin was purchased from GoldBio.

Lys6, Lys11, Lys29, Lys33, Lys48, Lys63, and linear linked di-Ub obtained from Boston Biochem were incubated at 10 μM with 20 nM BtSCoV-Rf1.2004 PLpro. Reactions were performed in AMC buffer at a volume of 45 μL and a temperature of 37°C. 10 μL samples were taken at the indicated time points and heat-shocked at 98°C for 5 min. Lys48 and Lys63 linked tetra-Ub obtained from Boston Biochem were incubated at 13.67 μM with 23 nM BtSCoV-Rf1.2004 PLpro. Reactions were performed in AMC buffer at a volume of 70 μL and a temperature of 37°C. 8 μL samples were taken at the indicated time points and heat-shocked at 98°C for 5 min. SDS-PAGE analysis was performed using Mini-PROTEAN TGX and Coomassie blue.