Fig. 1.

a Western blot analysis of LHX3 proteins extracted from transfected heterologous HEK293T cells with wild type and mutated pcDNA-LHX3a-myc construct. Immunodetection of LHX3a-myc was performed using an anti-myc mouse monoclonal antibody. Left panel, cytoplasmic extracts; right panel, nuclear extracts. b Western blot analysis to quantify the amount of each LHX3 protein (transfected nuclear extract) for electromobility shift assay experiments (EMSA). c EMSA was performed with a 2xCy5 LUEGO-based probe containing the LHX3-binding site of the alpha-GSU promoter. Supershift (SS) was performed with a monoclonal anti-6xHis antibody. S arrow indicates the position of the LHX3 DNA binding. P, Free probe; pcDNA, pcDNA empty vector; NS, non-specific band; 50-fold higher concentration unlabeled wild-type probe (same sequence as labeled probe) (50x comp WT), mutated probe used as a competitor (50x comp MUT). d EMSA was performed with decreasing (three-fold dilution series) amounts of each mutant LHX3 protein. WT LHX3: proteins from LHX3-WT-transfected HEK293T cells; p.(Leu196Pro): proteins from LHX3-p.(Leu196Pro)-transfected cells; p.(Arg208Gly): proteins from LHX3-p.(Arg208Gly)-transfected cells; p.(Arg310Pro): proteins from LHX3-p.(Arg310Pro)-transfected cells