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Fig. 7
Figure 7. Cytoplasmic SNRNP70 regulates the abundance of Z+AGRN splice variants (A) Agrin Z+ (Z+AGRN) isoforms identified by RT-PCR are shown schematically, with conserved residues between humans and zebrafish shown in red. Arrows depict the position of primers used for the qPCR analysis in (B)–(E). (B–E) RT-qPCR analysis in sibling and null animals as well as following ubiquitous overexpression of cytosolic hSNRNP70-eGFP using Tg(ubi:ERT2-Gal4; UAS:hSNRNP70ΔNLS3xNES-eGFP) embryos at 28 hpf. Primers were designed to specifically amplify the expression of non-z+agrn (agrn 33–34 (Δz)) isoforms (B), as well as the three z+agrn isoforms: 33-z8-34 (C), 33-z11-34 (D), and 33-z8-z11-34 (E). The graphs show mean values ± SEM for fold change in expression in relation to control animals (sib). ∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05; ns, not significant, one-way ANOVA, datapoints represent two independent biological replicates per group. (F) Representative images depicting a superficial layer of the trunk myotomes showing the distribution of α-BTX-labeled AChR clusters in mock- and Z+AGRN-treated sibling and null animals at 48 hpf. Images are lateral views with anterior to the left. Scale bars, 50 μm. (G) Quantification of the number of α-BTX clusters in the four groups. The graph shows mean values ± SEM. ∗∗∗∗p < 0.0001; ∗∗p < 0.01; ∗p < 0.05; ns, not significant, one-way ANOVA, n = 21 animals per group in two independent experiments. (H) Graph showing mean values ± SEM of the average size of α-BTX clusters in the four groups. ∗∗∗∗p < 0.0001; ∗p < 0.05; ns, not significant, one-way ANOVA, n = 21 animals per group in two independent experiments.