14-3-3s Induces Mouse ES Cell Proliferation Figure 5. 14-3-3s interacts with GSK-3b. (A) CCE, R1 and D3 cells transfected with control or 14-3-3s vectors were lysed and 14-3-3s complex was pulled down by immunoprecipitation (IP) with a Flag antibody. Proteins in the complex were analyzed by Western blotting (WB). (B) CCE cells were transfected with each isoform vector. 14-3-3 complex was pulled down and GSK-3b was determined by Western blotting. (C) Association of 14- 3-3s with WT or mutant GSK-3b was determined by immunoprecipitation (IP) with a Flag Antibody followed by Western blotting (WB) with GSK-3b and p-GSK-3b antibodies. (D) CCE cells transfected with Flag-tagged 14-3-3s (s) or control (CTR) vectors were treated with wortmannin. 14-3-3s complex was immunoprecipitated with a Flag antibody and GSK-3b was analyzed by Western blotting. (E) 14-3-3s overexpression reduced GSK-3b activity. Data represents mean 6 s.d. (n = 3). *, p,0.05; **, p,0.01. (F) CCE cells were transfected with control or 14-3-3s vectors for 42 h followed by treatment with wortmannin (Wort) for 6 h. Total and phosphorylated protein levels of GSK-3b and Akt were detected by Western blot analysis. doi:10.1371/journal.pone.0040193.g005 PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e40193