(A) EMSAs demonstrate that ARX-GST (220–392) can bind to double-stranded oligonucleotides that contain the ARX binding site; all mutations tested had the same binding activity as WT ARX (protein loading was carefully controlled to enable comparisons between constructs with 5 μg of protein loaded in each lane). (B–D) Quantification of the luciferase reporter assays in HEK 293T cells co-transfected with constructs as indicated (RLUs). (B) Ebf3-luciferase activity is significantly repressed by ARX (WT and mutant), with no significant difference between WT and mutant constructs transfection. (C) Cells were treated with indicated concentration of phosphatase inhibitor CA for 30 min. The treatment did not perturb the repressive transcriptional activity of ARX to Lmo1-luciferase activity. (D) Top-flash reporter assay indicated both WT and mutant ARX have the same ability to activate the Wnt signaling pathway (**P < 0.01, ***P < 0.001, ****P < 0.0001; n = 3). (E) Representative images of the embryonic cryosections from Arx^flox/flox Ai14^hom mice electroporated with Nestin:Arx^{WT or 8SA}-IRES-Cre. The cortex was divided into ten equal bins for the quantification. Cells electroporated with a WT construct or the 8SA construct showed a similar positional organization (n = 3 for each genotype).