4.10. Drug Affinity-Responsive Target Stability (DARTS)

The DARTS assay was performed according to a previously described protocol [58]. SK-HEP-1 cells were scraped and lysed in M-PER buffer (Pierce, Rockford, IL, USA) containing freshly added protease inhibitors for 10 min. After centrifugation at 18,000× g for 20 min at 4 °C, proteins were quantified via a BCA assay. The cell lysates or recombinant STAT3 protein (Δ127-722, 20 μg) was added to 10× TNC buffer (500 mM Tris-HCl (pH 8.0), 500 mM NaCl, 100 mM CaCl₂) and equally divided between two tubes for 1 h at 25 °C with DMSO or CIB-6. After digestion with a certain proportion of pronase (Roche, Basal, Switzerland) at 25 °C for 30 min, digestion was terminated by adding protease inhibitors. Aliquots of samples were mixed with 5× loading buffer and boiled for SDS-PAGE.