Fig. 6. Downstream signaling of ß-eng. Western blot analysis of three replicate HC11 cell cultures infected with ß-catenin, ß-eng or mock-infected control (A). Phospho-specific AKT antibody revealed no change in activated AKT (A, upper panel) compared with total AKT (A, lower panel). Quantification by densitometry (B) indicated that levels of pAKT relative to total AKT were not significantly changed as a consequence of the exogenous expression of ß-catenin or ß-eng. Semi-quantitative RT-PCR of target genes CD44 (C) and ITF-2 (E) at 18, 20 and 22 cycles of PCR (representative of three separate experiments). CD44 mRNA levels were increased slightly in HC11 cells infected with exogenous ß-catenin, whereas ITF-2 levels appeared unchanged. However, both CD44 and ITF-2 mRNA levels were decreased in cells expressing ß-eng (D,F). Quantitation of these results relative to the control L19 RNA showed that expression of ß-eng in HC11 cells downregulated the expression of both of these mRNAs by at least twofold.