Subcellular localization (A), DNA-binding (B) and transactivation activity (C) of the novel PITX3 mutation in comparison with the recurrent mutation. A. Immunocytochemistry of the PITX3 wild-type and mutant proteins transfected in B3 lens epithelial cells. Cells were stained with monoclonal anti-FLAG M2 primary antibody and Alexa Fluor 568 donkey anti-mouse IgG as a secondary antibody (red); Hoechst 33342 was used as a nuclear counterstain (blue). All three proteins localize predominantly to the nucleus. B. Results of EMSA experiments demonstrating the DNA-binding capacity of the PITX3 wild-type and mutant proteins. The profile of the wild-type PITX3 protein (third from the left) displays two major bands likely representing DNA interactions with PITX3 homo- or hetero-dimers (upper band; arrow) or PITX3 monomers (lower band; dashed arrow). The profile of the mutant PITX3 proteins only shows the lower band. Addition of anti-PITX3 antibody results in the appearance of a supershifted band (asterisk) and disappearance of the other bands. Western blot (bottom) shows similar levels of PITX3 wild-type and mutant proteins in the nuclear extracts used for EMSA experiments. C. Luciferase assay results for wild-type and mutant PITX3 co-transfected with the bcd-TK-luc or MIP656-bcd1,2-pGL3 reporters in human lens epithelial cells. The values are reported as fold changes of luciferase activity in comparison to an empty vector (pcDNA3.1). All luciferase activities were normalized to β-galactosidase activity and error bars indicate the standard deviation of two independent experiments performed in triplicate. The asterisk indicates the statistically significant differences in fold change (p < 0.001).