To quantify the effect of MRAP2 on the constitutive activity of GHSR1a, CHO-K1 cells were transfected with 67, 33, 17, 8.3, 4.2, 2.1, 1, 0.5, and 0 ng of GHSR1a per well with 0.9 μg of empty vector or MRAP2. Cells were lifted with TrypLE, resuspended in DMEM/F12, and plated at a density of 14,000 cells per well in a 384-well plate. The IP-One assay was performed as described earlier but omitting the agonist. Each condition was run in triplicate, and experiments were repeated independently at least three times.

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