orad Quantity One Gel Box. 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 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 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 25 mg mouse anti-HA antibody according to the manufacturer’s protocol. For immunoprecipitation 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 buffer. Then 5060 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. DNA plasmids The TKRlu plasmid with the Renilla luciferase gene under the control of the thymidine kinase promoter was used as a transfection control. The TCF-Luc plasmid, 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. The CRE-Luc plasmid, 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 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 gene 10411607” 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. Luciferase assay To measure the CRE reporter activity, HEK293 cells were transfected with CRE-Luc DNA. To measure the TCF reporter activity, either MedChemExpress JW-55 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 with 5% fetal bovine serum. Cells were plated on a 24-well plate for 24 h prior to transfection. Cells were transfected in Opti-MEM I with various plasmids10 ng of control TKRlu plasmid by use of Lipofectamine 2000 according to the manufacturer’s protocol. Cells were induced with the indicated reagents at 24 h after transfection. Seventeen hours 22473614” after induction, cells were harvested and lysed in the plate with 100 ml of 16 passive lysis buffer at room temperature for 15 min, and the firefly and Renilla luciferase activities were measured on an Envision luminometer with the Dual Luciferase assay kit. Firefly luciferase data were normalized to Renilla luciferase data. Cell lines, mice, and primary cell preparation HEK293, COS-1 and Hep3B cells were from ATCC. 293STF cells have been described. C57Bl/ 6J mice were purchased from The Jackson Laboratory. Mice were housed under specific pathogen-free conditions in
