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Drug affinity responsive target stability (DARTS)

HY Heng Yang
YL Yang Liu
MZ Mei-Mei Zhao
QG Qiang Guo
XZ Xi-Kang Zheng
DL Dan Liu
KZ Ke-Wu Zeng
PT Peng-Fei Tu
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Drug affinity responsive target stability assay (DARTS) was adapted from a previous study33. HepG2 cells were lysed in 350 μL lysis buffer (0.4% Triton X-100, 400 mM NaCl, 100 mM Tris-HCl, pH 7.5, 20% glycerol) supplemented with protease and phosphatase inhibitors. Lysates were diluted to the same final volume and incubated with bufalin (0, 2.5, 5, 10, and 20 nM) for 1 h at room temperature with shaking gently. Samples were then proteolyzed with pronase (2 μg/mL) for 10 min in reaction buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM CaCl2). Reactions were terminated by adding SDS-PAGE loading buffer and detected by immunoblot with a specific anti-SDC4 antibody.

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