assays were initiated by mixing 5 mM coelenterazine h (Renilla luciferase-specific substrate) (Biotium, Hayward, CA) with the cell suspension. The luminescence signals were collected immediately using a 2103 Envision fluorescence plate reader configured with the ,700 nm dichroic mirror and with dual emission filter sets for luminescence (460 nm, bandwidth 25 nm) and fluorescence (535 nm, bandwidth 25 nm). Fluorescence of the YFP was acquired by exciting the samples at 485 nm and collecting the emission at 525 nm. The BRET ratios were calculated based on the ratio of emission from YFP and Rlu, as described previously [33]. Saturation BRET studies were also performed as described previously [33]. In brief, COS-1 cells were transfected with a fixed concentration of Rlu-tagged constructs as donor (1.5 mg DNA/ dish) and with increasing concentrations of YFP-tagged constructs as acceptors (0.3 mg to 6 mg DNA/dish). After 48–72 h, BRET assays were performed. The BRET signals were plotted as ratios relative to the ratios of emissions of YFP/Rlu, and the curve fit was evaluated based on R2 values using Prism 4.0. (GraphPad, San Diego, CA). Supporting Information Figure S1 Lrp6 physically interacts with GCGR. A). HEK293 cells were transfected with a control vector (GFP), v5- tagged Lrp6, HA-tagged GCGR, or both for 24 h and then were treated with or without 50 nM GCG1-29 for 1 h. Cells were harvested and lysed and used for western blot analysis. The blot was probed with v5 antibody and then stripped and reprobed with HA antibody. The b-actin blot was used as a loading control. B). HEK293 cells were transfected and treated similarly as in A. The cells were harvested and lysed, and equal amounts of lysate were immunoprecipitated with the indicated antibody. For HA antibody, the antibody complex was pulled down by protein G beads. For v5 antibody, it was a single-step pull-down because the antibody was directly conjugated to the agarose beads. After pull- down, the beads were washed three times with 16TBST and then incubated in 16 SDS sample buffer to release the bound proteins. The lysates were used for western blot analysis and probed with the indicated antibody. (TIF) Figure S2 Coexpression of Lrp5 enhanced the CRE Luciferase activity. HEK293 cells were transfected with GCGR or GCGR+Lrp5 plasmids along with CRE-Luc and TKRlu (an internal control) on day 1. Cells were left untreated or treated with 50 nM GCG1-29 on day 2. Cells were harvested on day 3 to measure the luciferase activity as described. (TIF) References 1. Unson CG (2002) Molecular determinants of glucagon receptor signaling. Biopolymers 66: 218–235. 2. Li XC, Zhuo JL (2007) Targeting glucagon receptor signalling in treating metabolic syndrome and renal injury in Type 2 diabetes: theory versus promise. Clin Sci (Lond) 113: 183–193. 3. Wodarz A, Nusse R (1998) Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14: 59–88. 4. Huelsken J, Behrens J (2002) The Wnt signalling pathway. J Cell Sci 115: 3977–3978. 5. He X, Semenov M, Tamai K, Zeng X (2004) LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way. Development 131: 1663–1677. 6. Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20: 781–810. 7. Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, et al. (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423: 448–452. Glucagon Induced b-Catenin Signaling Pathway Figure S3 Sequence alignment of the intracellular loop 1 and 3, C-terminal region of Frizzled receptors and three class B GPCRs. The IC loops and C-terminal region were predicted by the HMMTOP server [35] and aligned by clustalW program [36]. The conserved residues critical for activation of Wnt/b-catenin signaling are highlighted in yellow based on previous studies [25]. Single mutations abolish Wnt/b- catenin signaling activity of human Frizzled 5 (Fz5) are indicated on the top of the alignment [25]. Residue number corresponds to human Fz5 sequence. (TIF) Figure S4 Effects of glucagon and Wnt3a on the TCF Luc reporter activity in the 293STF cells expressing GCGR and Lrp5. 293STF cells were transfected with 100 ng each of Lrp5 and GCGR plasmids and 10 ng of TKRlu (an internal control) on day 1. Cells were either left untreated, or treated with 50 nM GCG1-29, 10% Wnt3a Conditioned Media (CM) or 50 nM GCG1-29+10% Wnt3a CM on day 2. Cells were harvested on day 3 to measure the luciferase activity as described in the ‘‘materials and methods’’ section. The data label indicated the fold of induction compared with the non-treated group (NT). (TIF) Figure S5 The immunoprecipitation experiment using HA antibody. HEK293 cells were transfected with 2000 ng of the indicated DNAs and treated with or without 50 nM GCG1-29 for the indicated time, similarly as in Fig. 7. Cells were harvested and lysed, and cell lysates with equal amounts of protein were used for western blot analysis for Lane 1–3. For lane 4–10, the cells were harvested and lysed, and equal amounts of lysate were immunoprecipitated with HA antibody. The antibody complex was pulled down by protein G beads. After pull-down, the beads were washed three times with 16TBST and then incubated in 16 SDS sample buffer to release the bound proteins. The lysates were used for western blot analysis. (TIF) Acknowledgments We thank Jiandie Lin and Matthew Molusky at the University of Michigan for providing us with help in isolating primary liver cells from mice. We thank Alicja M. Ball and Mary-Lou Augustine for assistance with cell culture for the BRET studies. In addition, we thank David Nadziejka for editorial assistance in preparing the manuscript. Author Contributions Conceived and designed the experiments: JK CZ BOW HEX. Performed the experiments: JK CZ KGH CRZD LW LEM. Analyzed the data: JK CZ KGH LJM. Contributed reagents/materials/analysis tools: JK CZ CRZD KGH. Wrote the paper: JK KGH LJM BOW HEX. 8. Haq S, Michael A, Andreucci M, Bhattacharya K, Dotto P, et al. (2003) Stabilization of beta-catenin by a Wnt-independent mechanism regulates cardiomyocyte growth. Proc Natl Acad Sci U S A 100: 4610–4615. 9. Castellone MD, Teramoto H, Williams BO, Druey KM, Gutkind JS (2005) Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta- catenin signaling axis. Science 310: 1504–1510. 10. Kulkarni NH, Halladay DL, Miles RR, Gilbert LM, Frolik CA, et al. (2005) Effects of parathyroid hormone on Wnt signaling pathway in bone. J Cell Biochem 95: 1178–1190. 11. Suzuki A, Ozono K, Kubota T, Kondou H, Tachikawa K, et al. (2008) PTH/ cAMP/PKA signaling facilitates canonical Wnt signaling via inactivation of glycogen synthase kinase-3beta in osteoblastic Saos-2 cells. J Cell Biochem 104: 304–317. 12. Tobimatsu T, Kaji H, Sowa H, Naito J, Canaff L, et al. (2006) Parathyroid hormone increases beta-catenin levels through Smad3 in mouse osteoblastic cells. Endocrinology 147: 2583–2590. PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33676