Fig. 5

GFP rescue sensor assay.

A) Single cell embryos were injected with the GFP reporter (50 ng/μL) in the presence or absence of MO_miR-2188, miR-2188 and scrambled duplexes or a mixture of both MO_miR-2188 and miR-2188 duplex. Fluorescence levels were observed at 24 hpf and representative embryos of each condition were photographed. Fluorescence was recovered in MO_miR-2188+ miR-2188 injected embryos, when compared to miR-2188 duplex injected embryos. B) Embryo lysates were prepared from 24 hpf embryos injected with the GFP reporter in the presence or absence of MO_miR-2188, miR-2188 and scrambled duplexes or a mixture of MO_miR-2188 and miR-2188. Protein levels were determined by western blot analysis and β-tubulin was used as an internal control. C) Quantification of the reporter proteins (%). Asterisks indicate conditions where GFP expression was significantly down regulated by miR-2188 relative to control conditions. miR-2188 injected embryos showed a statistically significant down regulation of GFP levels, indicating that Nrp2a 3′UTR is a true miR-2188 target. Co-injection of the MO_miR-2188 with the miR-2188 duplex resulted in the rescue of GFP fluorescence, further confirming that Nrp2a is a bona fide target of miR-2188. Data are mean +/stdev, p<0,005 (t test, unpaired), n>3. All lanes were normalized to the β-tubulin signal.