Fig. 6. Sequence-specific Kaiso-mediated transcriptional repression is NLS dependent. (A) To determine the effects of the NLS point mutation on Kaiso transcriptional activity, wild-type and NLS-defective Kaiso effectors were cotransfected with the pGL3-4xKBS-luciferase reporter plasmid into HeLa cells. Endogenous Kaiso repressed transcription of the 4xKBS-luciferase reporter 1.6 times. Co-transfection of Kaiso further reduced luciferase activity fivefold (pGL3 + Kaiso), whereas NLS-defective Kaiso had a negligible effect (pGL3 + NLSmut) despite expression levels comparable to those of wild-type Kaiso, as determined by immunoprecipitation and immunoblot analysis in (B). The addition of one copy of the SV40-LTAg/NLS at the C-terminus of NLS-defective Kaiso fully restored Kaiso-mediated transcriptional repression of the luciferase reporter.