Fig. 4.

miR-K5 targets protein coding region of monocistronic ORF57 mRNA. (A) Genomic DNA expression constructs consist of a MYC tag and the region spanning ORF57 exons (E1 and E2), intron, and its entire 3′UTR (solid line between E2 and AATAAA). Defective miRNA binding sites are illustrated as small boxes within Exon 1 of MYC-57 tK5 (amino acid sequence encoded by tK5 is unchanged) and the intron of MYC-57 tK6-3p (tK5 and tK6-3p are described in Figs. 2 B and C). (B) Immunoblot analyses of MYC-57 and transfection control FLUC in HEK293 cells either mock transfected (-), or cotransfected with the indicated plasmids and miRNAs for 48 h. Bands labeled NS represent proteins that are nonspecifically cross-reactive with anti-MYC antibody. Quantitation of MYC-57/FLUC derived from three independent measurements is described and plotted in Fig. S4B.