Fig. 5

Two heterozygous transgenic zebrafish lines were generated by CRISPR/Cas9. A NEK9^78del protein is lacking the ATP-binding domain shown in the 3D structure of NEK9 (A). NEK9^500del is harboring a frame shift mutation leading to a premature stop codon. Sanger sequencing results of the affected allele are shown in Supplementary Fig. 5C. B, C Piscine ELC and mutated piscine myc-tagged NEK9 protein either lacking the ATP-binding domain (NEK9^78del) or harboring a premature stop codon (NEK9^500del) were overexpressed in HEK293 cells. The ELC-NEK9 interaction was analyzed by specific immunoprecipitation (IP) and immunoblot (IB) (B). Quantification of ELC-NEK9 interaction illustrated as averaged intensity of precipitated ELC protein normalized to ELC expression of input protein lysate (C). Data are mean ± SD (n = 3). *P < 0.05; ***P < 0.001 by the analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test. D, E Validation of ELC-NEK9 interaction. Human myc-tagged ELC protein and different deletion variants of human flag-tagged NEK9 protein were overexpressed and interaction intensity was quantified after IP and IB. Data are mean ± SD (n = 3). **P < 0.01 by the analysis of ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons test.