2.11. Drug affinity responsive target stability (DARTS) assay

The DARTS assay is often used for small molecule target identification and is based on the principle that proteins become less susceptible to proteases when they bind to small molecules/metabolites. We performed the DARTS assay following a previously published protocol [27]. One hippocampus was lysed in 400 μl M-PER (Thermo Fisher Scientific, 78503) supplemented with a protease inhibitor cocktail (Sigma Aldrich, Roche, 11836153001). The lysate was centrifuged (18,000 g, 15 min, 4 °C) and 360 μl supernatant was mixed with 40 μl 10× TNC buffer (10× Tris–HCl 0.5 M pH 8.0, NaCl 0.5 M, CaCl₂ 0.1 M). The lysate was further diluted with 400 μl 1× TNC buffer, which then gave a protein concentration of 2–5 mg/ml. The lysate was split into 50 μl aliquots, which were either treated with vehicle (double distilled H₂O) or G-1,6-BP at indicated concentrations. For complex formation lysates were incubated for 60 min on ice, followed by 15 min at room temperature. Thereafter pronase (Sigma Aldrich, Roche, 10165921001) or vehicle (double distilled H₂O) was added at indicated concentrations (mg pronase/mg protein lysate). Lysates were incubated for 30 min at 25 °C. The reaction was stopped by adding Laemmli buffer and incubating the lysates at 70 °C for 10 min. For lysates with low or high protease inhibitor cocktail treatment either vehicle (double distilled H₂O) or an additional protease inhibitor cocktail (Sigma Aldrich, Roche 11697498001) was added before treatment with G-1,6-BP. All other steps were performed as described above without the addition of pronase.