Glucagon Induced b-Catenin Signaling Pathway area represents the background signal of 0.12 determined using Rlu-tagged Lrp5 with soluble YFP as noted. Coexpression of untagged GCGR or Lrp5 competitively reduced the BRET signals obtained between Lrp5-Rlu and GCGR-YFP (black bars). Saturation BRET analysis supported the specific interaction of Lrp5 and GCGR. The non-specific bystander type BRET signal (linear) was observed when the non-structurally-related CCK1 receptors were co-expressed with Lrp5. Incubation with the natural agonist peptide ligand, glucagon (up to 1 mM), did not significantly change the Lrp5 and GCGR BRET signal. Data are represented as the means 6 S.E.M. from four to six independent experiments performed in triplicate. Data marked with ** were significantly different from background signals at the level of p,0.01. doi:10.1371/journal.pone.0033676.g007 Reagents LiCl was purchased from Sigma Aldrich. The antibodies used were mouse anti-b-catenin monoclonal antibody (BD Biosciences), mouse anti-b-actin antibody (Abcam), mouse anti-v5 antibody and v5 antibody-conjugated agarose beads (Sigma Aldrich), rabbit anti-GCGR antibody (H-57) (Santa Cruz), and mouse anti-HA antibody (Covance). Forskolin and H89 were obtained from Calbiochem. A Dual Luciferase assay kit was purchased from Promega. GLP-1 peptides, glucagon peptides, and PTH peptides were purchased from peptide 2.0. Dickkopf-1 (DKK1) protein was purchased from R&D systems. DNA plasmids The TKRlu plasmid with the Renilla luciferase (Rlu) gene under the control of the thymidine kinase promoter was used as a transfection control (Promega). The TCF-Luc plasmid (M50 Super 86 TOPFlash), with the firefly luciferase gene under the control of the TCF promoter, was obtained from Addgene. Mouse DKK1, Lrp5, Lrp6, and Wnt5-Fz8 plasmids have been described before [29,30]. The CRE-Luc plasmid (pGL4.29[luc2p/CRE/ Hygro]), with the firefly luciferase gene under the control of the CRE promoter, was obtained from Promega. The GCGR plasmid was obtained from Missouri S&T cDNA Resource Center. For immunoprecipitation, GCGR DNA was cloned into the pcDNA3.1 expression vector with three tandem HA tags at the C-terminus by PCR. The Lrp5 extracellular domain (ECD, residue 32-1375) was cloned into pcDNA6 vector with an Ig k leader sequence at the N-terminus to allow its secretion into media supernatant. For BRET studies, the yellow fluorescent protein (YFP) gene was cloned into the C-terminus of GCGR to create the GCGR-YFP fusion construct. The Rlu gene was cloned into the C-terminus of Lrp5 to create Lrp5-Rlu fusion construct. All constructs were verified by automated DNA sequencing. Cell lines, mice, and primary cell preparation HEK293, COS-1 and Hep3B cells were from ATCC. 293STF cells (which are HEK293 cells with an integrated ‘‘Super-Top- Flash’’ TCF-luciferase reporter) have been described [18]. C57Bl/ 6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed under specific pathogen-free conditions in micro-isolator cages under an American Association for Laboratory Animal Accreditation and Certification–approved protocol. Four- to six-week-old mice were used to isolate the primary hepatocytes. The procedures for isolating primary hepatocytes have been described previously [31]. Western blot analysis HEK293 or other cells were cultured in 6-well plates (Costar, Cambridge, MA). For experiments requiring transfection, cells were transfected with the indicated DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After transfection, cells were treated with different reagents for the indicated time. Cell pellets were lysed in 90 ml of 16 cell lysis buffer (Cell Signaling Technology) on ice for 30 min and western blots were performed as described earlier [32]. The blots were developed with Pico Chemiluminescence substrate (Pierce Bio- technology) and exposed to Kodak X-O mat films, which were scanned on an HP ScanJet flat-bed scanner (Hewlett Packard). Alternatively, the blots were exposed by using a Biorad Quantity One Gel Box (Biorad). For reprobing, membranes were stripped using a solution containing 62.5 mM Tris-HCl, 2% SDS, and 100 mM b-mercaptoethanol at 62uC for 45 min. Immunoprecipitation HEK293 cells in 6-well plate were transfected with HA-tagged GCGR with or without v5-tagged Lrp5 plasmids on day 1 with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. On day 2, cells were treated with or without GCG1-29 for 1 h. Cells were subsequently harvested and lysed in 100 ml of 16 cell lysis buffer (Cell Signaling Technology) on ice for 30 min. Cell debris was removed by centrifugation. The ExactaCruzTM E kit from Santa Cruz was used to immunoprecipitate HA-tagged GCGR from the cell lysate with 2–5 mg mouse anti-HA antibody according to the manufacturer’s protocol. For immunoprecipita- tion with v5 antibody, 50 ml of anti-v5 agarose beads were directly incubated with the cell lysate for 2 h at 4uC with mixing induced by rotation of the tube. The beads were spun down and washed three times with 16 TBST (Tris-buffered saline with 0.05% Tween 20) buffer. Then 50–60 ml of 26 reducing electrophoresis buffer was added to the beads and they were incubated at 37uC for 5 min before loading onto an SDS-gel for western blot analysis. Luciferase assay To measure the CRE reporter activity, HEK293 cells were transfected with CRE-Luc DNA. To measure the TCF reporter activity, either 293STF cells or HEK293 cells transfected with the TCF-Luc plasmid were used. HEK293 or 293STF cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 5% fetal bovine serum (FBS). Cells were plated on a 24-well plate for 24 h prior to transfection. Cells were transfected in Opti-MEM I (Invitrogen) with various plasmids+10 ng of control TKRlu plasmid (constitutive expression of Renilla luciferase) by use of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Cells were induced with the indicated reagents at 24 h after transfection. Seventeen hours after induction, cells were harvested and lysed in the plate with 100 ml of 16 passive lysis buffer (Promega) at room temperature for 15 min, and the firefly and Renilla luciferase activities were measured on an Envision luminometer (Perkin-Elmer) with the Dual Luciferase assay kit (Promega). Firefly luciferase data were normalized to Renilla luciferase data. BRET studies BRET experiments were performed using COS-1 cells as described previously [33]. Cells were seeded at a density of 0.56106 cells/dish in sterile 10-cm tissue culture dishes in DMEM supplemented with 5% Fetal Clone II (Logan, UT). After 24 h, the cells were transiently transfected with 3 mg of DNA/dish using the diethylaminoethyl (DEAE)-dextran method [34]. Cells were used 48–72 h later. Receptor-bearing COS-1 cell suspensions of approximately 25,000 cells/well were used for bioluminescence and fluorescence measurements in 96-well Optiplates. BRET PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e33676