modulated by b-catenin. PITX2 and b-catenin interact to increase the activity of PITX2 and co-expression of both activate the Dlx2 promoter at 30-fold whereas co-expression of Dact2 and b-catenin did not activate 
the Dlx2 promoter in LS-8 cells. The Dact2 expression plasmid was titrated against 19372201 constant amounts of PITX2 and b-catenin plasmids and the Dlx2 reporter. As the Dact2 plasmid concentration was increased from 0.5 mg, 1 mg, 2 mg, 4 mg and 8 mg, this correlated with increased MedChemExpress Digitoxin repression of PITX2 transcriptional activity in the presence of bcatenin. Dose dependent inhibitory effect of Dact2 also exists when b-catenin is not ectopically expressed. Thus, Dact2 inhibition of PITX2 is not b-catenin dependent and is not affected by the interaction between b-catenin and PITX2. Dact2 does not repress Dlx2 activation of the Dlx2 promoter indicating that the repression by Dact2 is specific to PITX2. To rule out the possibility that PITX2 was degraded after over expression of Dact2, Western blots were performed using PITX2A and Dact2 co-transfected LS-8 cells. Transfected PITX2A levels remained constant with different amounts of transfected Dact2. b-catenin over expression is shown by Western blot. TCF/LEF binding sites and the Fopflash plasmid contains point mutations in the TCF/LEF binding sites, which abolish binding of Lef-1 and was used as a negative control. Transfected Lef-1 activated the Topflash reporter by about 8-fold, and transfected Dact2 strongly repressed this activation. Co-transfection of PITX2A and Lef-1 activated Topflash at 90fold and addition of Dact2 repressed this 11478874 activation to,60-fold. Dact2 expression repressed the PITX2 and Lef-1 synergistic activation of Topflash by 30%. These results indicate that Dact2 represses the activation of tooth development factors and the Wnt/b-catenin pathway in general by interfering with PITX2 and Lef-1 transcriptional activity. This repression by Dact2 can be rescued in a dose dependent manner by introducing Dact2 shRNA. This result verifies that repression of Topflash is specificity due to Dact2 function. We noticed the mild activation on Fopflash by overexpressing Dact2. But this activation cannot be reversed by introducing Dact2 shRNA. Dact2 is both cytoplasmic and nuclear localized and colocalizes with Pitx2 in the nucleus The Dact1 protein shuttles between the nucleus and cytoplasm. Because Dact2 and Dact1 proteins have high homology, Dact2 may also have this property. We performed endogenous immunofluorescence staining on LS-8 cells. Dact2 labeled with FITC appears in both nuclear and cytoplasm compartments in discrete loci. Evidence from Xenopus showed xDact proteins are present in both cytoplasm and nuclear compartments. An IgG mock control staining was used in parallel to demonstrate the specificity of Dact2 staining. LS-8 cells transfected with Dact2 shRNA were also stained for Dact2 protein. Compared to non-transfected LS-8 cells and LS-8 cells transfected with scrambled shRNA , the overall level of Dact2 expression was significantly reduced by Dact2 shRNA, demonstrating the specificity of Dact2 staining. LS-8 cells express both Dact2 and Pitx2 and we used LS-8 cells to conduct endogenous immunoprecipitation assays. We were able to successfully pull down Dact2 proteins in the whole cell lysate by using a PITX2 antibody, compared to the IgG mock negative control. A GST-pull down assay was used to confirm the PITX2-Dact2 interaction. Dact2 protein and immobilized GST-PITX2A