Lys29

The substrate specificity of USP17 was tested against the di-ubiquitin chains of the eight linkage types (Lys⁶, Lys¹¹, Lys²⁹, Lys³³, Lys⁴⁸, or Lys⁶³ (Boston Biochem) and linear (LifeSenors)), as well as Lys¹¹(Boston Biochem), Lys⁴⁸, or Lys⁶³ (LifeSensors) tetra-ubiquitin chains. For reactions containing USP17 a concentration of 40 nM was used. Each di-ubiquitin reaction contained 4 μM of the ubiquitin chain and was incubated with or without enzyme at 25 °C for 120 minutes. The tetra-ubiquitin reactions contained 2 μM of the ubiquitin chains and were incubated with or without enzyme for a range of times from 10 to 120 minutes at 25 °C. Reactions were quenched with NuPAGE LDS sample buffer (Invitrogen) and analyzed by NuPAGE Novex 4-12% Bis-Tris mini gels (Invitrogen) stained with Coomassie Brilliant Blue.

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.