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Fluorometric Imaging Plate Reader (FLIPR) Analysis. Re-
ceptor assay was carried out by measuring the calcium flux in
response to the addition of agonist. HEK-EM 293 cells seeded in
96-well plates were loaded with Calcium 3 fluorescent calcium dye
(Molecular Devices, Sunnyvale, CA) for 1.5 h at room temperature
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measured on a FLIPR Tetra (Molecular Devices, Sunnyvale CA).
Measurements were taken every 0.5 s during the first 5 min of the
assay. Ten measurements were made before compound addition
followed by 450 after compound addition. Afterward, 10 more
measurements were taken for 5 s each. Experiments with each
mutant were carried out in parallel with the wild-type receptor in
triplicates or quadruplicates.
Data Analysis. Agonist-stimulated response in wild-type and
mutant receptors was taken as the maximum - minimum value by
subtracting the baseline response in vector-transfected control cells.
The dose-response relationship was analyzed by fitting sigmoidal
curves to the data sets using GraphPad Prism 4 (San Diego, CA).
The set of data obtained with the wild-type receptor and the mutant
within the same experiment were analyzed simultaneously to obtain
the relative EC50 values. The EC50 value for the wild-type receptor
was obtained as an average value from all experiments. The
response was normalized as percentages relative to that of the wild-
type receptor, and the data were presented as mean ( SEM of three
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Acknowledgment. This research was supported by the
Intramural Research Program of the National Institute of
Diabetes and Digestive and Kidney Diseases, NIH.
Supporting Information Available: Coordinates of the com-
plex GPR40-GW9508 in PDB format. Alignment of the sequences
of GPR40 from four different species and bovine rhodopsin. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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