Glucagon Induced b-Catenin Signaling Pathway Figure 1. Glucagon agonist activates the CRE-Luc activity in GCGR-expressing cells. A). Protein expression for GCGR in HEK293 cells and primary hepatocytes (isolated from BL/6 mice). HEK293 cells cultured in 6-well plate were transfected with 2000 ng of pcDNA3.1 (empty vector), GCGR or HA tagged GCGR for 24 h and cells were lysed for western blot analysis as described. B). HEK293 cells cultured in 24-well plate were transfected with pcDNA3.1 and CRE-Luc (50 ng each) or CRE-Luc and GCGR plasmids (50 ng each) along with 5 ng TKRlu (an internal control) on day 1; cells were treated with or without the glucagon agonist GCG1-29 (50 nM), the antagonist GCG9-29 (50 nM) or forskolin (FSK, 10 mM) on day 2, and were harvested for luciferase activity measurements after about 17 h on day 3. Triplicate samples were used for each treatment. In all experiments, the CRE promoter–driven firefly luciferase activity was normalized to Renilla luciferase activity driven by the thymidine kinase promoter (for transfection control). Luc activity for non-treated group was set to 1 and Luc activities for treated groups were adjusted accordingly. *p,0.005 compared with the non-treated group. C). Primary hepatocytes cultured in 12-well plates were transfected with CRE-Luc (400 ng) and TKRlu (10 ng) plasmids on day 1, then treated with or without 50 nM GCG1-29 on day 2 and harvested on day 3 for luciferase activity measurement. Duplicate samples were used for each treatment. *p,0.001 compared with the non-treated group. doi:10.1371/journal.pone.0033676.g001 PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33676