Phosphorylation of 14.3.3ζ at serine 58 requires up-regulation of Akt1 protein levels. (A) The effect of increasing concentrations of Akt1 on phosphorylation of 14.3.3ζ was analyzed in cell lysates of SW480 cells. Akt1, 14.3.3ζ, p14.3.3ζ, and pS6 Rib were analyzed by Western blot. Actin was used as a loading control. (B) The expression of Akt1 and Akt2 after IFNγ treatment was analyzed by Western blotting lysates of colonic tissue of mice injected with IFNγ. Western blots were performed using antibodies against pan-Akt, Akt1, Akt2, pAkt308, and pβ-cat552. Actin was used as loading control. (C) The role of Akt1 expression in β-catenin transactivation was evaluated in SW480 cells transfected with 0.01–2 μg/ml vector expressing Akt1. β-Catenin transactivation was evaluated by TOPflash assay. TOP vs. FOP and TOP/FOP activity are plotted. (D) Effects of Akt1 subcellular distribution on β-catenin–mediated transcriptional activity was evaluated by TOPflash assay in SW480 cells. Cells were transfected with 0.75 μg/ml Akt1-myr, Akt1, or Akt-NLS constructs in serum-free medium. (E) The expression of p14.3.3ζ was analyzed in colonic cell lysates of IFNγ-injected animals that were pretreated with dimethyl sulfoxide or Akt inhibitor VIII. Densitometric values were normalized to actin and are presented as a graph (**p < 0.0001).