Drug affinity responsive target stability assay

A drug affinity responsive target stability (DARTS) assay for identifying the targets of SZB120 were performed according to the protocol by Lomenick et al. (29, 30). In brief, Jurkat cells or T cells in Th17 cell differentiation conditions were lysed, and protein concentrations were determined by the BCA Protein Assay Kit (Thermo Fisher Scientific) to ensure an equal amount of protein lysate per sample. Cell lysates were treated either with various concentrations of SZB120 (1–100 μM) or with DMSO alone. After incubation overnight at 4°C, digestion was performed using Pronase (Roche). Each cellular lysate sample was proteolyzed at room temperature for 10 min with 0.05 mg/ml Pronase and stopped using excess protease inhibitors. The samples were then loaded on SDS-PAGE gels and stained with Silver Stain (Thermo Fisher Scientific). Finally, the bands with increased staining from the SZB120 lane and the matching area of the control lane were cut out, digested with in-gel trypsin, and subjected to liquid chromatography-tandem mass spectrometry analysis.