Figure 2.

C3G reduces β-catenin protein and expression of β-catenin/TCF target genes. (a) Effect of C3G on exogenously expressed β-catenin protein levels. HEK 293T cells transfected with 200 ng of β-catenin expression vector and 20 ng of GFP along with 200 ng of pcDNA, C3G-GFP, or GFP-CBR expression vectors were lysed after 30 hours of expression and subjected to Western blotting with the indicated antibodies. TfR is a β-catenin target gene. GFP and actin were used as transfection and loading controls, respectively. Numbers indicate decrease in β-catenin and TfR levels in C3G expressing cells relative to levels in control plasmid transfected cells. (b) Effect of C3G expression on cellular β-catenin protein. MDA-MB-231 cells were transfected with 400 ng of C3G expression vector, and cell lysates made after 48 hours of expression were subjected to Western blotting, along with lysates from untransfected cells (UT), with the indicated antibodies. Numbers indicate quantitation as in (a). (c) HEK 293T cells were transfected with 200 ng of β-catenin and 200 ng of pcDNA or C3G and 20 ng of GFP vectors. Cells were treated with 40 µg/mL of cycloheximide after 10 hours of expression for the indicated time periods. Cell lysates made at each time point were subject to Western blotting with the indicated antibodies. Tubulin was used as loading control. (d) CBR domain of C3G enhances turnover of endogenous β-catenin. MDA-MB-231 cells transfected with 400 ng of GFP or GFP-CBR were treated with 40 µg/mL of cycloheximide after 30 hours of expression for the indicated time periods. Cell lysates made at each time point were subject to Western blotting with the indicated antibodies.