HEK293 cells were seeded in 24-well plates coated with poly-l-lysine (Sigma-Aldrich) at 100,000 cells per well and transfected with 375 ng of plasmid coding for the wild-type or mutant CysLT1R using X-tremeGENE HP (Roche). Forty-eight hours after transfection, cells were fixed with 3.7% (v/v) formaldehyde in tris-buffered saline [TBS; 20 mM tris-HCl (pH 7.5) and 150 mM NaCl] for 5 min at RT. Cells were washed three times with TBS and incubated for 1 hour in TBS supplemented with 3% fat-free milk (w/v) to block nonspecific binding sites. A mouse monoclonal anti-HA antibody coupled to horseradish peroxidase (Roche) was added at 1:1000 dilution in TBS with 3% fat-free dry milk for 3 hours at RT. Following incubation, cells were washed twice with TBS before the addition of 250 μl of 3,3′,5,5′-tetramethylbenzidine (Sigma-Aldrich). Plates were incubated at RT for 15 min, and the reaction was stopped by the addition of 250 μl of 2N HCl. The reagent (200 μl) was transferred into a 96-well plate, and the absorbance was read at 450 nm on a Tecan Genios Pro plate reader. Cells transfected with the empty pcDNA3.1(+) vector were used to determine the background. Data were plotted using GraphPad Prism 7 and represent the means ± SD of at least two independent experiments performed in quadruplicate.

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