Fig. 6.

miR-494 targets the NCL 3′UTR. (A) Schematic of the detection of microRNAs interacting with an RNA of interest. HeLa cells were transfected with the plasmids indicated in Fig. 5B. (B) Forty-eight hours after transfection of plasmids, RNP IP analysis was performed using anti-EGFP antibody, and the levels of microRNAs present in the IP samples were assessed by RT-qPCR analysis. The microRNAs enriched greater than 2-fold in the MS2-RL-NCL(3′) mRNA IP group compared with those in the MS2-RL mRNA IP group are shown. (C) Forty-eight hours after transfection of miR-494 or an antisense miR-494 antagomir [(AS)miR-494], the levels of nucleolin and loading control β-actin were assessed by Western blot analysis and quantified. (D) Forty-eight hours after transfection with either Ctrl siRNA or miR-494, the enrichment of NCL mRNA in HuR IP samples was tested by RNP IP followed by RT-qPCR analysis. Data in panels B to D are the means and standard deviations (s.d.) from three experiments. *, P < 0.05.