Drug affinity responsive target stability (DARTS) assay

The DARTS assay was performed according to the protocol previously described [32]. HaCaT cells were treated with 1,8-cineole or DMSO plated in 10-cm dishes at 80% confluency, before the cells were lysed after 1 hour in M-PER buffer (Pierce, Rockford, IL, USA) containing protease and phosphatase inhibitors. After centrifugation (12,000 rpm, 10 min, 4°C), 10x TNC buffer [500mM Tris·HCl (pH 8.0), 500mM NaCl, 100 mM CaCl₂] was added to the lysates, before the protein concentration was determined with a dye-binding protein assay kit (Bio-Rad Laboratories) following the manufacturer's instructions. The lysates (2.5 μg/ μL) were digested with pronase (1: 3,000 or 1: 10,000 of protein to pronase ratio) for 30 min. Digestion was stopped by adding 5x Laemmli sample buffer, before the samples were subjected to Western blot analysis.