Fig. 4. NLS-dependent nuclear translocation of full-length Kaiso. (A) To validate the relevance of the identified Kaiso NLS in the context of full-length Kaiso, wild-type and NLS-defective Kaiso were fused N-terminally to eGFP. Consistent with the localization of endogenous Kaiso, eGFP/Kaiso was targeted predominantly to HeLa-cell and Va-2-cell nuclei, as determined by confocal microscopy. NLS-defective Kaiso with a K472A point mutation demonstrated a nearly exclusive cytosolic localization in all cell lines tested. (B) To verify that the eGFP/Kaiso fusion protein was intact and that the eGFP moiety had not been cleaved, we counterstained transfected cells with our Kaiso-specific antibodies (red) and visualized the stained cells on an Axiovert 200 inverted microscope. Our antibodies efficiently and specifically detected the eGFP/Kaiso fusion proteins, indicating that the nuclear fluorescence was due to intact eGFP/Kaiso fusion proteins. (C) To confirm the expression of intact eGFP/Kaiso and eGFP/Kaiso-NLSmut proteins of correct molecular weight, we performed immunoprecipitation and immunoblot analysis of whole-cell lysates from transfected cells, using specific anti-GFP antibodies. The eGFP/Kaiso and eGFP/Kaiso-NLSmut fusion proteins migrated at the expected molecular weight of 125 kDa, whereas eGFP alone migrated at 30 kDa. Bars, 20 µm.