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Fig. 3 miR-30 directly targets the 3′UTR of the Hedgehog transmembrane receptor smoothened.
(A–F) Embryos injected with 3 different GFP reporter mRNAs; (A,B) the GFP ORF plus tandem perfect target sites (GFP-PTS), (C,D) GFP ORF plus the smo 32UTR sequence (GFP-SMO) and (E,F) the GFP ORF without UTR sequence (GFP- no UTR). Constructs were injected either alone (A,C,E) or with the miR-30 duplex RNA (B,D,F). (G) Western blot validation on lysates of GFP injected embryos with and without the miR-30 duplex (H) Densitometric analysis of GFP protein levels normalised against α-tubulin loading control shows a 54% reduction in GFP-SMO+miR-30 compared to GFP-SMO only embryos. (I) Protein blot analysis of smoothened levels in wild type and miR-30 morpholino knockdown embryos shows an increased level of Smoothened protein. (J) Densitometric analysis of the average change in smoothened protein level in 3 samples of wild type versus miR-30 morpholino treated embryos.