A20 supports β-catenin ubiquitination and degradation through an interaction with the destruction box.

<p>(A) Luciferase assay showing transcriptional activity of a β-catenin dependent TCF/LEF4 reporter in RKO cells. Cells were treated with A20 specific or control siRNA and recombinant human wnt3a (rhwnt3a) as indicated. Relative luciferase units (RLU) are shown. **indicates p<0.01. (B) Co-precipitation of A20 with Axin. RKO cells transfected with the indicated expression plasmids were lysed, immunoprecipiated (IP) for the indicated epitope tag, and immunoblotted (IB) for the indicated proteins. Cells were stimulated with rhwnt3a or control as indicated for four hours. Input levels of MYC, FLAG, and GAPDH are shown as controls below. (C) Co-precipitation of partial A20 proteins with Axin. Co-transfection experiments as in (B). Input levels of MYC, FLAG, and GAPDH shown below. (D) A20 suppresses wnt3a stimulated induction of β-catenin expression. Immunoblot analyses of active and total β-catenin expression in RKO cells treated with A20 specific or control siRNA. A20 and GAPDH levels shown below as loading control. (E) A20 supports wnt3a stimulated β-catenin ubiquitination. RKO cells were treated with A20 specific or control siRNAs and wnt3a for the indicated times. Lysates were immunoprecipitated for β-catenin followed by immunoblotting for ubiquitin. Input amounts of beta-catenin, A20, and GAPDH proteins shown below as controls. All data are representative of three or more independent experiments.