TAMRA-VME Probe Assay for USP19 Activity

The TAMRA-VME probe assay was carried out as described previously [40 (link)]. Briefly, HEK293T cells transfected with USP19-CY-wt, USP19-CY-CA, USP19-ER-wt or USP19-ER-CS were lysed in TAMRA ABP buffer (50 mM Tris–HCl, pH 7.4, 250 mM sucrose, 5 mM MgCl₂, 1 mM DTT, 0.5% zwitterionic surfactant CHAPS and 0.1% nonyl phenoxypolyethoxylethanol (NP40) supplemented with protease inhibitors. Then, the samples were sonicated for five cycles of 30 s (s) on and 30 s off on ice. Thereafter, the cell lysates were centrifuged at 16 × 10³ g for 15 min at 4 °C, and the supernatants were transferred to fresh Eppendorf tubes to measure the protein concentrations. The carboxytetramethylrhodamine ubiquitin-vinyl methyl ester (TAMRA-Ub-VME) probe (UbiQ-050; UbiQ) was used at a concentration of 1 µM to label 25 µg of protein extracts in a total volume of 25 µL for 30 min at room temperature. The labeling reactions were terminated by the addition of sample buffer and heating to 100 °C for 10 min. The labeled proteins were separated by NuPAGE 4–12% Bis–Tris protein gels (WG1402BOX; Invitrogen), and the fluorescence signals were detected using the Typhoon FLA 9500 Molecular Imager (GE Healthcare) at an excitation wavelength of 550 nm and an emission wavelength of 590 nm.