Fig. 2.

Responses of controls in the 384-well formatted GnRHR[E⁹⁰K] and wild type pharmacoperone assay. (A) Mutant cells. In the absence of doxycycline (“Dox”), GnRHR[E⁹⁰K] is synthesized and retained in the endoplasmic reticulum (ER). Following pretreatment with the pharmacoperone IN3, GnRHR[E⁹⁰K] is rescued and trafficked to the plasma membrane, and a robust Ca²⁺ response to GNRH (500 nM) challenge is observed (●). Also included are the results of challenging the cells with GnRH, after preincubation with IN3 and doxycycline at 1 μg/mL (▲), as well as similar experiments without the GnRH challenge (■ and ▼). (B) Wild type cells. In the absence of Dox, wild type GnRHR is expressed and automatically trafficked to the plasma membrane. Upon GnRH challenge, the pharmacoperone IN3 decreases the amount of Ca²⁺ response in these cells, in a concentration-dependent manner, because IN3 acts as an antagonist in this format (●). Also shown are the control experiments in the wild type cells, similar to those done with the mutant cells. The data presented are means±standard deviation (sd) of quadruplicate wells (n=4).