UXT is evolutionarily conserved and developmentally expressed. (A) A sequence alignment of human, mouse, rat and zebrafish UXT proteins by Clustal X. The histogram below the ruler indicates the degree of similarity. Peaks indicate positions of high similarity and valleys indicate low similarity. The asterisk indicates positions that have been fully conserved. The red rectangles indicate the conserved amino acid sites for UXT-2M mutation. (B) Whole-mount in situ hybridization of UXT in zebrafish embryos from 6hpf to 72hpf. The developmental time points are indicated in the panels. (C) Quantification of the UXT mRNA expression in zebrafish embryos from 6hpf to 72hpf by using RT-qPCR. Data show the mean±s.e.m. (at least three independent experiments). (D) Antibody staining of UXT in zebrafish embryos from 6hpf to 72hpf. (E) Quantification of UXT protein expression in zebrafish embryos with the UXT-specific antibody 4B4, from 0hpf to 72hpf, normalized to GAPDH. Densitometry was performed using ImageJ. Data show the mean±s.e.m. (at least three independent experiments). (F) Phenotypic analyses of zebrafish embryos at 24hpf. Embryos were injected with 4ng of control morpholino (Ctrl MO), 4ng of UXT MO2 or 4ng of UXT MO2 with 150pg of UXT mRNA (UXT MO2+UXT mRNA). Additional embryos were injected with 4ng of UXT MO2 plus 150pg of UXT mRNA mutant (UXT MO2+UXT-2M mRNA). All embryos were analyzed at 24hpf. The morphological defects are indicated by black arrowheads. (G) The knockdown efficiency of the morpholinos. Embryos were injected with 4ng of control morpholino, 4ng of UXT MO1, 4ng of UXT MO2 or 4ng of UXT MO3. The embryos were harvested at 24hpf and the lysates were probed with the anti-zebrafish UXT-specific antibody 4B4. (H) Graphical representation of zebrafish phenotypic analyses; the number of embryos analyzed in each group is indicated above the bars.
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