Fig 5

miR-1 directly targeted sec63. (A) Subcellular localization of potentially targeted proteins differentially expressed in miR-1 morphants and negative control embryos identified by gene ontology annotations. 9 proteins were upregulated and 23 proteins were downregulated in miR-1 morphants. (B) Protein expression of Sec63 in the control and miR-1 MO-injected groups. (C) A candidate protein for upregulation, sec63, was determined by iTRAQ analysis. Validation of increased sec63 expression in miR-1 morphants by qPCR in vivo at 24 hpf. Data are expressed as the mean ± SD, from at least three independent experiments, n=15, **P < 0.01. (D) Expression of sec63 in embryos at 24 hpf. Embryos were viewed laterally, with the anterior to the left. sec63-3ʹUTR binding site of miR-1 was predicted using TargetScan (E, F). (G) Mutant 3ʹUTR of sec63 in dual luciferase reporter plasmids. Overexpression of miR-1 (miR-1-pre) reduced sec63-3ʹUTR luciferase activity in vitro, but not the luciferase activity of mutated sec63-3ʹUTR. Three independent experiments were performed, and each experiment was carried out in duplicate (H).