Figure 3. eIF5A1 is a target mRNA of miR-434-3p. (A) Sequence alignment of putative miR-434-3p targeting site in the 3’-UTR of eIF5A1 shows high levels of complementarity. (B-D) Myotubes were transfected with miR-434-3p mimetic or NS-mimetic. miR-434-3p overexpression was determined by qPCR assay (B). U6 served as a normalizer. eIF5A1 mRNA levels were analyzed 24 h after transfection by qPCR (C) and eIF5A1 protein levels were analyzed 36 h after transfection by Western blot (D). (E) Sequence information represents the site directed mutagenesis on the 3’UTR of eIF5A1. Red letters indicate mutated nucleotide and arrows indicate nucleotide position. (F) The eIF5A1 3’-UTR–luciferase construct or eIF5A1 3’-UTR mutated–luciferase construct were co-transfected with NS-mimetic, miR-434-3p mimetic, miR-434-3p antagomir or SC-miR. Forty-eight hours after transfection, cells were collected, and then firefly luciferase activities were estimated and normalized to Renilla luciferase activities. Each bar indicates mean ± SEM (n = 3). *P < 0.05 vs. NS-miR. Gel pictures are representative of three independent experiments.