The prepared membrane homogenates (50 μl) were plated in triplicate in poly-d-lysine–coated plates and spun at 2500g for 10 min at 4°C. One hundred microliters of a solution of 2% (w/v) glutaraldehyde in PBS was added and incubated 15 min at room temperature. Supernatant was removed, and 100 μl of 0.1 M glycine and 0.1% BSA in PBS was added to each well for 30 min at room temperature. Liquid was aspirated and wells were blocked in 5% (w/v) milk in PBS for 30 min at room temperature. Anti-HA antibody (1:2000) in 5% (w/v) milk was added to wells and incubated for 1.5 hours at room temperature. Wells were washed three times in PBS before adding secondary antibody (anti-mouse HRP, 1:5000) in 5% (w/v) milk for 45 min at room temperature. Wells were washed three times in PBS, and 50 μl of substrate (3,3′,5,5′-tetramethylbenzidine; Sigma, T0440) was added for 5 min at room temperature. Reactions were terminated by adding 50 μl of 10% (v/v) sulfuric acid, and absorbance at 450 nm was measured on a SpectraMax i3 plate reader (Molecular Devices). Results from GTPγS binding assays were normalized to GHSR1a levels for each sample.

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