Fig. 5. NLS-defective p120^ctn does not inhibit Kaiso-mediated transcriptional repression. (A) Artificial promoter assays in HeLa cells revealed that endogenous Kaiso repressed luciferase expression from the 4x KBS-luciferase construct approximately twofold (compare pGL3 Control to 4x KBS). Overexpression of wild-type Kaiso (4x KBS + Kaiso) repressed luciferase expression an additional twofold. Overexpression of wild-type p120^ctn inhibited Kaiso-mediated repression of the luciferase reporter, whereas the NLS-defective p120^ctn mutant (NLS Mut) failed to inhibit Kaiso-mediated transcriptional repression despite similar expression levels to wild-type p120^ctn, as shown in (B). Expression levels of transfected p120^ctn were detected by immunoprecipitation with p120^ctn-specific antibody 8D11, which recognizes exogenous mouse, but not endogenous human, p120^ctn. Inhibition of Kaiso-mediated transcriptional repression by p120^ctn was fully rescued by fusing the SV40-LTAg NLS to the carboxy-terminus of NLS-defective p120^ctn (compare 4x KBS + NLS Mut with 4x KBS + NLS Mut + SV40 NLS). Migration of p120^ctn fused C-terminally to the SV40-LTAg NLS was impeded slightly on SDS-PAGE due to the addition of the heterologous NLS. Similar results were also obtained in Cos-1 cells (data not shown).