Fig. 3: Structure-guided point mutations in SOX2 disrupt binding to IMPα3.

a Mutations within SOX2 were introduced based on the interaction interface and were tested in pull-down assays. His-tagged SOX2 constructs was immobilized on Ni²⁺ agarose beads in the presence of IMPα3 (see input), washed, and eluted through TEV cleavage. Three single-point mutations disrupted binding: K42A, R43A, and K115A. A single SOX2 triple mutant incorporating three mutations was confirmed to not interact with IMPα3 in the equivalent pull-down experiment. See also Supplementary Fig. 6 for uncropped gels. Results were reproduced independently in three separate experiments with similar results. b The effect on binding was also assessed in MST assays, where SOX2 was shown to bind with 102 ± 15 nm (n = 3, where n represents three independent experiments) affinity, whereas no binding of SOX2x3Mut (red) could be detected. The data points are presented as mean values ± standard deviation.