Supplemental Data
Supplemental Data
Files in this Data Supplement:
- Supplemental Data - Supplementary Figure 1. (A) Lack of GPR39-dependent β-arrestin recruitment response to obestatin in TANGO assay with 100 μM ZnCl2 or without ZnCl2. This experiment was performed in quadruplicate. (B) GPCRome β-arrestin recruitment in response to obestatin as measured by the TANGO assay, performed using 1 μΜ obestatin with 100 μM ZnCl2. The results are the mean ? S.E.M of quadruplicate determinations. PC (blue); Positive control, 1 μM quinpirole at DRD2. Supplementary Figure 2. Concentration-response curves showing β-arrestin recruitment responses to two GPR39 agonists to non-GPR39 targets as measured in the TANGO assay. (A) GPR39-C3 (middle panels) stimulates a response at HTR1A, but not at other 5-HT1 subfamily receptors; LY2784544 (right panels) is inactive at all 5-HT1 subfamily receptors. Positive controls with 5-HT are shown in the left panels. The bottom panel shows that 5-HT is inactive at GPR39. (B) LY2784544 (right panel) stimulates a response at MTLNR1A melatonin receptors, but GPR39-C3 (middle panel) does not; positive control with melatonin is shown in the left panel, and the bottom panel shows that melatonin is inactive at GPR39. Blue points and curves show experiments done in the presence of 100 μM ZnCl2, and red points and curves show experiments done in the absence of ZnCl2. Supplementary Figure 3. Antagonist-mode β-arrestin recruitment measured using the TANGO assay, showing that various concentrations of LY2784544 do not block GPR39- mediated responses to GPR39-C3, done in the absence of Zn2+. Values were normalized using the GPR39-C3 response as 0% (basal) to 100% (Emax). This experiment was performed in quadruplicate. Supplementary Figure 4. Cell surface expression levels of GPR39 constructs and mutants in HEK293T cells. (A) TANGO constructs and (B) ?deTANGOized? constructs. Cell surface expression levels were measured by Cell ELISA (methods). The results are the mean ? S.E.M. of at three independent experiments performed in quadruplicate. Supplementary Figure 5. Ca2+ influx response to GPR39-C3 (A), LY2784544 (B) or GSK2636771 (C) with various concentrations of ZnCl2 in HT-29 cells. The results are the mean ? S.E.M. of at least three independent experiments performed in quadruplicate. Supplementary Figure 6. Ca2+ influx response to GPR39-C3 (A), LY2784544 (B) or GSK2636771 (C) with 100 μM ZnCl2 or without ZnCl2 in PC-3 cells. The results are the mean ? S.E.M. of at two independent experiments performed in quadruplicate.