14-3-3s Induces Mouse ES Cell Proliferation Figure 4. 14-3-3s overexpression enhances b-catenin stability and nuclear translocation. (A) CCE cells were transfected with control (CTR) or 14-3-3s vectors for 48 h. Phosphorylated b-catenin (p-b-catenin) was determined by Western blot analysis. Actin was used as loading control. (B) b-catenin ubiquitination was analyzed by immunoprecipitation (IP) followed by Western blotting (WB). (C) Cell lysate and nuclear fraction were prepared from CTR and 14-3-3s-transfected CCE cells and b-catenin was determined by Western blot analysis. LaminA/C and Actin were used as the loading control of nuclear and cytosolic fractions. (D) b-catenin transactivation activity was determined by luciferase-based TOP reporter assay. FOP reporter activity was used as a control. Data represent mean 6 s.d. (n = 3). **, p,0.01. (E) Cyclin D1 expression in CTR or 14-3-3s transfected CCE was determined by Western blot analysis. Actin was used as loading control. doi:10.1371/journal.pone.0040193.g004 and sequestration of phosphorylated GSK-3b in a PI-3K/Akt dependent manner. 14-3-3s Overexpression Disrupts Association of b- catenin with the Multiprotein Destruction Complex Since 14-3-3s overexpression induces GSK-3b binding and inactivation, we reasoned that 14-3-3s overexpression could reduce b-catenin association with GSK-3b in the multiprotein destruction complex. To assess this, CCE cells were co-transfected with/without HA-tagged b-catenin and 14-3-3s vectors. Associ- ation of b-catenin with APC, axin or GSK-3b was analyzed by immunoprecipitation followed by immunoblotting. Overexpres- sion of 14-3-3s significantly reduced association of b-catenin with GSK-3b as well as APC and axin, and increased the b-catenin level (Figure 6A). Besides, the binding of overexpression or endogenous b-catenin with p-GSK-3b is barely detected (Figure 6A). In addition, transfection of S9A GSK-3b mutant abolished 14-3-3s-induced b-catenin expression while transfection of T309A did not (Figure 6B). These results are consistent with the interpretation that 14-3-3s level is pivotal in controlling GSK-3b action and regulating b-catenin bioavailability. To provide additional evidence for this, we evaluated the effect of RA on pGSK-3b and b-catenin levels. RA treatment suppressed 14-3-3s- increased pGSK-3b and reduced p-b-catenin (Figure 6C). 14-3-3s transfection restored pGSK-3b and increased b-catenin reduced by RA (Figure 6C). Furthermore, RA treatment for 48 h slightly reduced Oct3/4 level. However, 14-3-3s did not significantly affect Oct3/4 expression (Figure 6C). These results indicate that PLoS ONE | www.plosone.org 6 June 2012 | Volume 7 | Issue 6 | e40193