Pathhunter detection kit 93 0001

β-arrestin2 recruitment downstream of ACKR3 activation was analyzed using the PathHunter CHO-K1 CXCR7 β-arrestin Cell Line (93-0248C2; DiscoverX). Cells were cultured in AssayComplete™ Cell Culture Kit-107 (92-3107G; DiscoverX) at 37 °C and 5% CO₂. Briefly, 20,000 cells/well were seeded in AssayComplete™ Cell Plating 2 Reagent (93-0563R2A; DiscoverX) in a white 96-well plate (100 µL/well) with clear bottom (Costar, 3610) and incubated overnight at 37 °C and 5% CO₂. The next day, 10 µL/well of test compound (11 × concentrated) was added and cells were incubated for 90 min at 37 °C and 5% CO₂. Compounds were tested in duplicate in a 1:4 dilution series ranging from 1000 nM to 0.06 nM final concentration. After incubation, 55 µL/well of detection reagent (PathHunter Detection kit; 93-0001; DiscoverX) was added and cells were incubated for 1 h at room temperature (RT) protected from light. Finally, luminescence was measured using a FLIPR Tetra^® device (Molecular Devices, Sunnyvale, CA, USA). Four parameter non-linear curve fitting (GraphPad Prism 9.0.2) was used to determine the EC₅₀ value for β-arrestin2 recruitment.

BDNF (PHC7074) and ToxBLAzer reagent (K1136, P36400) were obtained from Life Technologies. NGF-β (N1408), NT-3 (N1905), NT-4 (N1780), and Thapsigargin (T9033) were obtained from Sigma-Aldrich. Tool small molecules: LM22A-4, amitryptiline, 7,8-dihydroxyflavone and N-acetyl serotonin were supplied by CHDI and used after passing QC analysis; B_AG peptide was synthesized by Peptide Synthetics; tool antibody panel was sourced commercially (BD mAb #610102; Millipore pAb #07-225) or from Pfizer Inc (38B8 and 29D7 mAbs); a pan-Trk kinase inhibitor was synthesized at BioFocus [26] . All-trans retinoic acid (R2625) and BSA (A0281) were obtained from Sigma-Aldrich. PathHunter® detection kit (93-0001) was obtained from DiscoveRx. DMSO (D/4121/PB17), Triton-X100 (10102913) and Halt Protease and Phosphatase Inhibitor Cocktail (100X) (78444) were obtained from ThermoFisher Scientific. Recombinant pAKT1 (31145) for Meso Scale Discovery studies was obtained from Active Motif; whole cell lysate kit – Phospho (Ser473)/ Total AKT assay (K11100D-2) was obtained from Meso Scale Discovery.