Binding was measured by a competition binding assay using isolated membranes from GLP1R-HEK293 cells. Membranes were incubated for 2.5 hours at room temperature in Hanks’ balanced salt solution (HBSS), 20 mM Hepes, 0.1% BSA (pH 7.4), 0.1% BSA with 0.075 nM 125I-GLP1 (7–36) (PerkinElmer #NEX308) as a tracer in the absence or presence of compounds over a range of concentrations. Following incubation, membranes were filtered through a filter plate (Millipore, #MSFCNXB50) and washed with ice-cold 25 mM Hepes, 1.5 mM CaCl2, 1 mM MgCl2, 100 mM NaCl, and 0.1% BSA (pH 7.4) to separate bound from unbound radioligand and subsequently quantitated by scintillation counting (MicroBeta TriLux, PerkinElmer).

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