Functional characterization of two mutant LHX4.

<p><i>A</i>, Protein expression level of myc-tagged WT and two LHX4 mutants was assessed by western blot using a monoclonal anti-myc antibody. The expression of V75I LHX4 was comparable to that of WT, whereas R84X LHX4 was not detected. Tubulin was used as a control. <i>B</i>, Subcellular localization analysis. For subcellular localization analyses, we visualized and photographed COS7 cells transfected with GFP-tagged LHX4 using a Leica TCS-SP5 laser scanning confocal microscope, after mounting the cells in Vectashield-DAPI solution. The WT and V75I LHX4 are localized to the nucleus. <i>C</i>, EMSA experiments. WT LHX4 showed specific binding to the elements, which was competed by excess amount of (200 times) cold competitors. The V75I LHX4, which has an intact HD, bound with similar or slightly high efficiency to the WT LHX4.