Fig. 6

miR-27 targets FAK and regulates FAK levels in vivo. (A) Schematic of the reporter mRNA consisting of the coding sequence of GFP fused to the ptk2aa 3′UTR. Two predicted miRNA recognition elements (MREs) are indicated. Predicted base-pairing between MREs (shown in green) and the miR-27a sequence (shown in red). (B) Embryos injected with either GFP-ptk2aa 3′UTR reporter mRNA alone or co-injected with miR-27a imaged at 24hpf. (C) Reporter assays with GFP-ptk2aa 3′UTR mRNA containing mutations in the miR-27 seed sites. (D) Western blots with anti-GFP and anti-tubulin antibodies using the lysates from embryos injected with the GFP-ptk2aa 3′UTR reporter mRNA alone or co-injected with miR-27a. (E) Quantification of GFP levels by Western blots normalized to the levels of tubulin. (F) Western blots with anti-FAK and anti-GAPDH antibodies using lysates from 24hpf embryos either uninjected or injected with 100 or 200 pg miR-27a. (G) Western blots with anti-FAK and anti-GAPDH antibodies using lysates from 48hpf embryos injected either with MO-ctl or MO-27. (H) Quantification of FAK levels from Western blots shown in (G) normalized to the levels of tubulin. At least 20 embryos were pooled for protein lysates. Error bars indicate SEM. **p<0.01 (Student's t-test).