Figure 7. Mutation of the miR-378 binding sites inhibits nephronectin glycosylation.

(A) Mutation was generated in the 3′UTR of Npnt+3′ construct as shown (Npnt+3′-mu, upper panel). This impaired both putative binding sites for miR-378 (bottom). (B) Strategy of PCR showing interaction of miR-378 with Npnt 3′UTR, which was abolished by sited-directed mutagenesis. (C) Typical PCR products are shown using the mature miR-378 as a primer for the PCR (arrow). The lower bands were primers used for the PCR assays. (D) Cell lysate prepared from cells transfected with Npnt+3′ and the mutant Npnt+3′-mu was analyzed with western blot showing decreased expression of the mutant construct.