Glucagon Induced b-Catenin Signaling Pathway Figure 7. Lrp5 physically interacts with GCGR. A). HEK293 cells cultured in 6-well plate were transfected with 2000 ng of a control vector (GFP), v5-tagged Lrp5, 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 equal amounts of protein were used for western blot analysis. The blot was first 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 same 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 16SDS sample buffer to release the bound proteins. The lysates were used for western blot analysis and probed with the indicated antibody. C–E). BRET data for Rlu-tagged Lrp5 and YFP-tagged GCGR expressed on COS- 1 cells. Shown are the static BRET signals (C), saturation BRET analysis (D), and effect of natural agonist ligand binding on the BRET signals (E). Shaded PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33676