Key cAMP Secondary GPR119 Assay HEK293 GPR119 Expression HEK293 Control 6 Cell Line Control (DCEA 9 0 0.010.1110100 0.010.1110100 Concentration(uM) Concentration(uM) Extended Data Figure 5. Secondary assay of GPrllg ACTOne HEK293 cells(control)and ACTOne heK 293 cells transfected with GPR119 were exposed to equimolar concentrations of the endogenous GPrI19 ligand oleoylethanolamide or the bacterial ligand N-oleoyl serinol. Relative fluorescent intensity was recorded for each ligand concentration compared to background signal. All data points were performed in quadruplicate and error bars represent SD around the mean. An increase in cAMP concentration was observed in HEK293 cells expressing GPr119 but not in native HEK293 cells. The DCEa [5-(N-Ethylcarboxamido)adenosine] control is presented to confirm cAMP response of the parental cell line. The EC50 for N-oleoyl serinol(bacterial) was 1.6 HM and for oleoylethanolamide was 5.1 HM, which are consistent with data from the B-arrestin assay(Figure 5a) Nature. Author manuscript; available in PMC 2018 February 28Extended Data Figure 5. Secondary assay of GPR119 ACTOne HEK293 cells (control) and ACTOne HEK293 cells transfected with GPR119 were exposed to equimolar concentrations of the endogenous GPR119 ligand oleoylethanolamide or the bacterial ligand N-oleoyl serinol. Relative fluorescent intensity was recorded for each ligand concentration compared to background signal. All data points were performed in quadruplicate and error bars represent SD around the mean. An increase in cAMP concentration was observed in HEK293 cells expressing GPR119 but not in native HEK293 cells. The DCEA [5–(N-Ethylcarboxamido)adenosine] control is presented to confirm cAMP response of the parental cell line. The EC50 for N-oleoyl serinol (bacterial) was 1.6 µM and for oleoylethanolamide was 5.1 µM, which are consistent with data from the β-arrestin assay (Figure 5a). Cohen et al. Page 19 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript