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Fig. 4 KR mutants repress AP-2α function
(A) AP-2α activity in a reporter assay [19] was inhibited by the K252R and 4xKR mutants with similar efficacy as the WT protein, although inhibition was reduced at low concentration. Immunoblots with anti-Kctd15, anti-AP-2 and anti-Tubulin antibodies control for protein expression. (B) AP2-Luc reporter assay using human KCTD15 and K278R mutant, both of which inhibit activity. (C) Kctd15 and K252R mRNAs were injected into zebrafish embryos. ISH with foxD3 probe at the 1 somite stage shows that the K252R mutant inhibits NC formation as effectively as WT Kctd15. Embryo images define normal expression, total inhibition and partial inhibition. Quantification is shown in the histogram, numbers of embryos listed on the bars. (D) Luciferase assay using AP-2γ illustrates that zebrafish WT and K252R mutant are equally effective in inhibiting reporter activity.