14-3-3s Induces Mouse ES Cell Proliferation Figure 6. 14-3-3s reduces b-catenin association with the APC/axin/GSK-3b complex and increases b-catenin. (A) Association of HA- tagged (left panels) and endogenous (right panels) b-catenin with the APC complex was determined by IP with HA antibodies and Western blotting (WB) with the indicated antibodies. (B) CCE cells were transfected with GSK-3b WT or the indicated mutants and b-catenin, GSK-3b, APC and Axin proteins were analyzed by Western blotting. (C) CCE cells with or without 14-3-3s overexpression were treated with RA. The indicated GSK-3b, b- catenin and Oct3/4 proteins were analyzed by Western blotting. (D) Cells were treated with or without RA for 48 h, the association of endogenous 14- 3-3s with GSK-3b was determined by IP with GSK-3b antibody and Western blotting of 14-3-3s. doi:10.1371/journal.pone.0040193.g006 14-3-3s may regulate mouse ES cell proliferation without influence stemness and pluripotency. To further evaluate the endogenous association of 14-3-3s with GSK-3b, we performed the IP experiments and found that GSK-3b interacts with endogenous 14-3-3s (Figure 6D). Treatment of RA significantly reduced 14-3-3s binding with GSK-3b (Figure 6D). Taken together, these results indicate high 14-3-3s levels increase b- catenin by releasing b-catenin from GSK-3b and confer resistance to RA-induced b-catenin degradation by maintaining GSK-3b phosphorylation. 14-3-3s Acts in Concert with Wnt to Control GSK-3b As anticipated, Wnt3a increased pGSK-3b and b-catenin in CCE cells (Figure 7A). 14-3-3s overexpression augmented Wnt3a- induced pGSK-3b phosphorylation and b-catenin elevation (Figure 7A). DKK1, an inhibitor of Wnt signaling blocked the action of Wnt3a on pGSK-3b and b-catenin but had a lesser effect on 14-3-3s transfected CCE cells (Figure 7A). We next de- termined the GSK-3b binding by 14-3-3s in the presence of Wnt3a and 14-3-3s transfection. In the absence of 14-3-3s overexpression, we detected lower interaction between endoge- PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e40193