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Fig. S3 The in vitro and in vivo analyses for the specificity of puf-A MO1. (A) Various amounts of puf-A-5mmMO1 (5 bp mismatch control: 0, 2, and 200 nM) were added to the in vitro transcription/translation reactions for puf-A. One microliter of the reaction mixture was separated on 10% SDS/PAGE, blotted and incubated with streptavidin-AP, followed by development with NBT-BCIP reagents.(B) For in vivo experiment, the puf-A 5′-UTR and its partial coding region were added onto pEYFP-N1 plasmid which contained CMV promoter and YFP gene to generate ppuf-A-YFP. Then 4.6 ng of the puf-A-MO1 or puf-A-5mmMO1 was co-injected to embryo with ppuf-A-YFP plasmid at 100 pg per embryo. The numbers of embryo with YFP expression were enumerated at 1dpf. As shown, 15/48 embryos injected puf-A-YFP plasmid exhibited fluorescence at 100 pg/embryo dosage, while none of the 113 embryos co-injected with the puf-A-MO1 had YFP expression. In contrast, co-injection with the mismatch control, puf-A-5mmMO1, did not affect the expression of YFP. (C) The panels showed the phenotypes of WT and morphants at 3dpf after injection with 9.2 ng of puf-A-5mmMO1 dosage.