(A, B) Dose-dependent inhibition for forskolin-stimulated cAMP accumulation. HEK 293 cells expressing SSTR2 or SSTR3 were exposed to 25 µM forskolin plus SST-14, (SST14)2-HSA or (SST28)2-HSA in concentrations ranging from 10−12 to 10−5 M for 15 minutes. Cells were lysed and cAMP levels were determined using a cAMP ELISA kit. The maximum forskolin-stimulated cAMP formation in the absence of agonist was defined as 100%. Values represent the means ± S.E. of three separate measurements performed in triplicate. The half-maximal inhibitory concentrations (IC50) were analyzed by nonlinear regression curve fitting using GraphPad Prism 5.0. (C, D) Time-dependent inhibition for forskolin-stimulated cAMP accumulation. HEK 293 cells expressing SSTR2 or SSTR3 were pre-treated with 1 µM SST-14, (SST14)2-HSA or (SST28)2-HSA, at 0.5, 1, 2, 4, 6, 8 hour time points, cells were exposed to 25 µM forskolin for 15 minutes. Cells were lysed and cAMP levels were determined using a cAMP ELISA kit. The maximum forskolin-stimulated cAMP formation in the absence of agonist was defined as 100%. Values represent the means ± S.E. of three separate measurements performed in triplicate.