FFAR1 Receptor Binding Assay

FFAR1 was cloned into the expression vector pCMV-Tag 2B encoding a N-terminal FLAG tag epitope (Stratagene), using the QuikChange method. COS7 cells were transfected using calcium phosphate precipitation method. IP accumulation assays were conducted as described by Hauge et al. (7 (link)). Competition binding assays were performed by adding increasing doses of TAK-875 and Compound 1 to the binding buffer (Hepes wash buffer + 100 µg/mL bacitracin), and immediately after, 50 µL tracer solution containing ³H-TAK-875 (5,000 cpm/well) was added. Plates were incubated for 3 h and washed twice, and γ-radiation was counted on a Packard Top Count NXT counter. Additional details are included in the SI Appendix.