Drug Affinity Responsive Target Stability (DARTS) assay and Mass
spectrometry (MS)
Approximately 1×107 of untreated RA FLSs were lysed
with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific)
containing protease and phosphatase inhibitors by shaking on ice for 20
minutes. 10× TNC buffer ( 1 M Tris-HCl 5 ml, 5 M NaCl 1 ml, 1 M
CaCl2 1 ml, double distilled H2O 3 ml,
PH 7.4) was added into the cell lysate (500 μg total protein, 5 mg/mL)
to reach the indicated concentrations and gently mixed. AZM and the
protein lysate were incubated for 1 hour at room temperature to allow
the target protein and ligand to sufficiently bind, and then digested by
pronase (Roche, dissolved in 1× TNC buffer) at a 1:1000 ratio (wt/wt)
for precisely 15 min at room temperature. After drug incubation and
protease digestion of protein cleavage products, 5× protein loading
buffer was added and boiled for 5 minutes. Then SDS-PAGE was performed,
and the bands were visualized by Coomassie blue staining. Finally, the
gel of bands with obvious changes were identified by mass spectrometry
(BGI, China) (Online Supplementary Table 3).