Figure 3.

Impact of the p.Y259N mutation on SOX17 mRNA and protein levels. A: Steady-state levels of SOX17 protein are profoundly affected by the p.Y259N mutation. A total of 100, 200, or 300 ng of WT SOX17 or p.Y259N SOX17 expression plasmids were transfected into HEK293T cells and protein lysates were harvested after 48 hr and analyzed by Western blot with an anti-SOX17 antibody, as well as an antitubulin antibody to assess protein loading. Comparison of SOX17-WT and SOX17-p.Y259N protein levels at equal dosage of transfecting DNA (lanes 2 and 5, 3 and 6, 4 and 7) suggests that the mutation increases SOX17 protein stability. Experiment was repeated four times and a representative blot is shown. B: SOX17 mRNA levels measured by quantitative RT-PCR. SOX17 mRNA levels in mock-transfected cells were 0.003 times that measured in cells transfected with 10 ng WT SOX17 (data not shown). N=3; ^*P=0.001; Error bars represent SEM. C: The effect of the p.Y259N mutation on the transcriptional activity of a SOX17 reporter plasmid. SOX17 expression plasmids were cotransfected with a SOX17-luciferase reporter plasmid. The SOX17-p.Y259N protein showed enhanced activity of the reporter at every transfection dosage, relative to the SOX17-WT protein. Data are shown as ratio of activities of firefly luciferase to control renilla luciferase. N=3; ^*P<0.05; Error bars represent SEM. D: The effect of the p.Y259N mutation on the ability of SOX17 to repress Wnt signaling. SOX17 expression plasmids were cotransfected with a TCF-luciferase reporter plasmid (TOP-flash) and an expression plasmid encoding a stabilized form of β-catenin. Repression of Wnt signaling was increased at every dosage with the mutant SOX17 protein, compared to the WT protein. Data are shown as ratio of activities of firefly luciferase to control renilla luciferase. N=3; ^*P<0.05; Error bars represent SEM.