JNK2 Interacts with b-Catenin nology, Lake Placid, NY), followed by immunoblotting probed with anti-HA (for b-catenin) or anti-GSK3b (for GSK3b). Mammalian two-Hybridization Assay HEK293T cells were plated at a density of 2.56105 cells/well in 24-well plate the day before transfection. Two mg of pACT- JNK2, pBind-b-catenin and pG5luc were co-transfected using Lipofectamine 2000 (Invitrogen, CA). pACT and pBind were used as negative controls. To eliminate non-specific interactions, two groups of negative controls were set: pACT-JNK2 and pBind, pACT and pBind-b-catenin. After 48 h, samples were lysed using 1x Passive lysis buffer, and the amount of firefly luciferase and Renilla luciferase was quantified using the Dual-Luciferase Reporter Assay System (Promega Corporation, Masison, WI), as we described recently [15]. The experiments were triplicated independently. Immunofluorescence Staining As described recently [2], HEK293T cells were co-transfected with pEGFP-b-catenin and pcDNA3-Flag-MKK7-JNK2. Twen- ty-four h after transfection, cells were fixed and stained with anti- Flag antibody (1ﬂ200; Genescript, Piscataway, NJ), followed by incubating with tetramethyl rhodamine isothiocyanate (TRITC) labeled anti-mouse IgG (Santa Cruz Biotechnology), mounted with UltraCruz 49-6-Diamidino-2-phenylindole (DAPI) containing mounting medium (Santa Cruz Biotechnology), visualized under a Zeiss LSM510 META confocal microscope (Zeiss, Jena, Ger- many). Images were taken and analyzed using Zeiss LSM Image Browser. Results and Discussion Activated JNK2 Suppressed Wnt/b-catenin Expression and Transcriptional Acticcity The studies from us and others have demonstrated that JNK1 can antagonize the canonical Wnt/b-catenin signaling [2,4]. To elucidate the potential role of JNK2 in the regulation of Wnt/b- catenin signaling, constitutively active JNK2 (MKK7-JNK2) was co-transfected with b-catenin into HEK293T cells. As shown in figure 1A (Lane 3 versus lane 1), b-catenin protein level was Figure 3. Active JNK2-mediated b-catenin degradation occurred through the proteasome system and GSK3b. (A) HEK293T cells were co-transfected with pcDNA3-HA-b-catenin and pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Forty-four hours after transfection, 25 mM MG132 was added to the indicated samples (lane 2 and 4). Four hours later cells were harvested for immunoblotting analysis to detect the expression of HA-b-catenin and p-JNK. (B) Blocking GSK3b activity by LiCl reduced b-catenin expression inhibition by activated JNK2. pcDNA3-HA-b-catenin was transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Thirty- six hours after transfection, half of the cultures were treated overnight with 30 mM LiCl (lane 2 and 4) and then harvested for immunoblotting analysis to detect the expression of HA-b-catenin, phospho-Ser-9 GSK3b, and p-JNK. (C) Mutant b-catenin was resistant to activated JNK2 induced degradation. Wild-type b-catenin (HA- b-catenin) (lanes 1 and 2) or various b-catenin mutants (HA-S33F b-catenin, lanes 3 and 4; HA-S33Y b-catenin, lanes 5 and 6; HA-S37A b-catenin, lanes 7 and 8) were transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 2,4,6,8) or empty vector (lanes 1,3,5,7). 48 hours after transfection, cells were harvested for immunoblotting analysis to determine the protein levels of HA-b-catenin. b- actin served as loading control. doi:10.1371/journal.pone.0006640.g003 PLoS ONE | www.plosone.org 3 August 2009 | Volume 4 | Issue 8 | e6640