(Madison, WI). For transfection, cells were plated to form a 50– 70% confluent culture. The HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen Corp.). For the A549 cells, transfection was carried out by Lipofectamine combined with Plus reagent (Invitrogen Corp.). Immunoblotting Standard immunoblotting analysis was used as described [2]. Blots were probed with the antibodies specific for HA, p-JNK, p-c- Jun, b-catenin (Santa Cruz Biotechnology), GSK3b, p-GSK3b (Cell Signaling Technology, Beverly, MA) and b-actin (Sigma– JNK2 Interacts with b-Catenin Aldrich Corp.) and the bands were visualized using enhanced chemiluminescence plus reagents (Amersham Pharmacia, Piscat- away, NJ). TCF-4 Reporter Assay Cells were co-transfected with pcDNA3-HA-b-catenin and pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, along with TOPFLASH or FOPFLASH. 24 or 48 h after transfection, the cells were harvested to conduct a firefly and Renilla luciferase activity assay using a dual luciferase kit (Promega). The TCF-4 reporter activity was presented as by ratio of firefly to Renilla luciferase activity. Each experiment was triplicated independently. Mouse Intestinal Epithelial Cell Isolation and Immunoblotting Similar as we described previously [16], intestinal epithelial cells were isolated from JNK2+/+ and JNK2-/- mice [17] small intestine by incubating with 15 mM EDTA buffer. Resulting cell pellets were lysed for immunoblotting for JNK2, b-catenin, GSK3b, p-GSK3b, CDK4 (Santa Cruz Biotechnology, Santa Cruz, CA) and b-actin analysis. Immunoprecipitation As described [16], whole-cell lysates were incubated with specific anti-Flag antibody to pull down MKK7-JNK2 using the Catch and Release reversible Immunoprecipitation system (Upstate Biotech- Figure 1. Active JNK2 downregulated b-catenin expression, inhibited its transcriptional activity and reduced GSK3b phosphorylation. (A) Active JNK2 suppressed b-catenin expression and GSK3b phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-b-catenin together with pcDNA3-Flag- MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after trans- fection, cells were harvested for immunoblotting analysis to detect the alterations of HA-b-catenin, p-JNK, p-c-Jun, phospho-Ser9 GSK3b, and GSK3b. b-actin served as loading control. (B) Active JNK2 reduced GSK3b phosphorylation and downregulated b-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-b-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of b-catenin, p-JNK, and phospho-Ser9 GSK3b. b- actin served as loading control. (C) Active JNK inhibited b-catenin- mediated transcriptional activity of TCF. HEK293T cells were co- transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7- JNK2, pcDNA3-HA-b-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean 6 standard deviation (SD) for triplicated samples. doi:10.1371/journal.pone.0006640.g001 Figure 2. Active JNK2 downregulated b-catenin expression and inhibited its transcriptional activity in a dose-dependent manner. (A) Activated JNK2 reduced b-catenin protein level in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-b-catenin along with different amounts of pcDNA3-Flag- MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA- b-catenin and p-JNK. b-actin served as loading control. (B) Activated JNK2 inhibited b-catenin-mediated transcriptional activity of TCF in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-b-catenin, TOPFLASH, Renilla, along with different amounts of pcDNA3-Flag-MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean 6 standard deviation (SD) for triplicated samples. doi:10.1371/journal.pone.0006640.g002 PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6640