14-3-3s Induces Mouse ES Cell Proliferation Figure 1. 14-3-3s is involved in mESC proliferation. (A) CCE cells were treated with 10 mg/mL retinoic acid (RA) for 12 to 72 h as indicated, and protein levels of 14-3-3 isoforms were determined by Western blot analysis. Actin was used as loading control. (B) CCE cells were transfected with scramble or 14-3-3s siRNA and each 14-3-3s isoform protein was determined by Western blot analysis after 48 h transfection. (C) Viable CCE cells were determined by trypan blue assay, and (D) BrdU incorporation was analyzed. Error bars are mean 6 s.d. (n = 3). N.S. denotes statistically not significant; *, p,0.05. doi:10.1371/journal.pone.0040193.g001 counted by using a hemocytometer. Cell proliferation was analyzed with a bromodeoxyuridine (BrdU) cell proliferation assay kit (Chemicon). Briefly, BrdU, a thymidine analog, is incorporated into newly synthesized DNA as cells enter the S phase. Following partial denaturation of double-stranded DNA, BrdU was detected immunochemically with a specific mouse monoclonal antibody. The amount of BrdU was determined after the addition of IgG-peroxidase conjugated secondary antibody, peroxidase substrate and stop solution. Promoter Activity Assay b-catenin promoter activity was measured by using TOP- FLASH/FOPFLASH reporter (Millipore). TOPFLASH/FOP- FLASH constructs and 14-3-3s or control vectors were incubated with Effectene transfection reagent in a 12-well plate for 48 h. Cells were washed with PBS and lysed in lysis buffer (Promega). Luciferase activity was measured with Luciferase Assay Reagent (Promega), and the emitted light was determined in a luminometer. Immunoprecipitation and Ubiquitination Assay CCE cells were transfected with 14-3-3s-Flag or co-transfected with b-catenin and/or GSK-3b for 48 h. Cells were harvested and lysed in RIPA buffer for 30 min at 4uC. After centrifugation, cell lysates were immunoprecipitated with a mouse monoclonal anti- Flag, anti-HA antibodies or mouse IgG as a control. The immunoprecipitates were resuspended in Laemmli sample buffer with 2-mercaptoethanol and boiled for 15 min. Proteins in the immunoprecipitate were separated by SDS–PAGE and analyzed by immunoblotting with rabbit polyclonal antibodies against b- catenin, GSK-3b, axin or APC. To evaluate the isoform-specific interaction of 14-3-3 with GSK-3b, each Flag-tagged 14-3-3 isoform expression vector was transfected into CCE cells, immunoprecipitated with an anti-Flag antibody, then immuno- blotted with anti-GSK-3b antibody. To investigate the phosphor- ylated residue of GSK-3b that is involved in interaction with 14-3- 3s, GSK-3b wild-type (WT), GSK-3b S9A mutant or GSK-3b T309A mutant constructs were co-transfected with 14-3-3s-Flag vector. Transfected CCE cells were immunoprecipitated with anti- Flag antibody, then immunoblotted with anti-GSK-3b antibody. For assay of b-catenin ubiquitination, CCE cells were co- transfected with 14-3-3s-Flag or HA–b-catenin expression vectors PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e40193