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. 2012 Oct;82(4):575–582. doi: 10.1124/mol.112.080358

Fig. 3.

Fig. 3.

Second-messenger studies performed with MIN6 cells. A, B, calcium assays. MIN6 cells transfected with the Rq or Rq(R165L) constructs (A) or vector DNA (pcDNA) (B) were incubated with increasing concentrations of CNO or OXO-M, respectively. Drug-induced changes in [Ca2+]i were measured by via FLIPR. A, data from a representative experiment (two additional experiments gave similar results). B, pooled data from three independent experiments. All assays were performed in duplicate. The CNO EC50 value at the Rq receptor was 28.2 ± 7.7 nM, and the OXO-M EC50 value at the wild-type M3 receptor was 1.00 ± 0.12 μM, respectively (means ± S.E.M., n = 3). AU, arbitrary units. B, cAMP assay. MIN6 cells transiently expressing the Rq(R165L) construct were incubated with increasing concentrations of forskolin or CNO. cAMP concentrations were measured in cell lysates by using a fluorescence resonance energy transfer-based cAMP kit (for details, see Materials and Methods). Data are given as means ± S.E.M. of three independent experiments, each performed in duplicate.