Ez link biocytin

BLI assay was used to detect the kinetics and affinities of compounds 8 and 19 to USP28. The EZ-Link NHS-LC-LC-biotin (Thermo Fisher Scientific, Waltham, MA, USA, Catalog#: 21343) was used for biotinylating USP28 protein at a molar coupling ratio (MCR) of 1:1. After incubation for 30 min at room temperature, the extra biotins were removed by the Slide-A-Lyzer™ G2 Dialysis Cassette (Thermo Fisher Scientific, Catalog#: 87734). The protein concentration of each sample was diluted to 5–10 μg/mL with PBST (PBS containing 0.02% Tween 20) buffer, and the biotinylated protein further linked to the superstreptavidin biosensors (FortéBio Inc., Shanghai, China) at 30 °C for 5 min. A curing test was performed to evaluate the quality of the protein biotin label, and the blank control set of sensors were activated in buffer (25 mmol/L Tris pH 7.5, 150 mmol/L NaCl and 0.05% DMSO). Finally, the sites of unbound biotin sensors were re-blocked with 5 μmol/biocytin (EZ-Link Biocytin, Thermo Fisher Scientific, Catalog#: 28022) for 1 min at 30 °C. Data analysis was a double reference subtraction (sample and sensor references) using the FortéBio data analysis software.

The BLItz system (Pall ForteBio) was used for BLI interaction assays. Short RNAs obtained by IVT as described above were biotinylated using Pierce RNA 3′ End Biotinylation Kit according to the manufacturer's instructions (Thermo). Short biotin-labeled RNAs containing different 5′ ends were immobilized onto streptavidin-coated biosensors (Pall ForteBio) by immersing the sensor in 1 µM RNA solution in kinetic buffer (50 mM phosphate buffer pH 7.2, containing 150 mM NaCl, 10% glycerol, 0.5 mM DTT, 0.1% BSA, and 0.05% Tween 20) for 5 min with 1000 rpm shaking. To minimize nonspecific interactions the sensor was blocked with 10 µg/mL EZ-LINK Biocytin (Thermo) and washed with kinetic buffer. The association of the proteins was measured by incubating RNA immobilized sensors in various concentrations of IFIT1 or IFIT5 proteins (2–1000 nM) diluted in kinetic buffer. The dissociation constants were measured by transferring the biosensor from protein solution to kinetic buffer and incubated for 5 min. The ForteBio analysis software was used to fit and analyze the data. The mean values of equilibrium dissociation constant K_D, and kinetic association and dissociation rates k_a and k_d were calculated from three independent experiments.