Figure 3.

1 is a competitive antagonist of TSH-stimulated signaling. A, HEK-EM 293 cells stably expressing TSHRs were exposed to the noted concentrations of 1 20 min before the addition of a half-maximally effective concentration of TSH (1.8 nm, control) and 1 mm IBMX. After 40 min incubation with 1 and TSH, the cells were lysed and cAMP levels were measured by ELISA. B, HEK-EM 293 cells stably expressing TSHRs were incubated in the absence of 1 or in the presence of 3, 10, or 30 μm 1 that was added 20 min before the addition of increasing concentrations of TSH (0.18 nm to 1.8 μm) and 1 mm IBMX. After 40 min, the cells were lysed and cAMP levels were measured by ELISA. The data from two independent experiments with duplicate samples are shown as percentage of maximum. A Schild plot of these data was linear with a slope not different from 1.0 (not shown). bTSH, Bovine TSH.