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. 2009 Oct 22;38(1):239–252. doi: 10.1093/nar/gkp870

Figure 3.

Figure 3.

Flanking sequence and 3′UTR context effects on miR-223 mediated inhibition. (a) Sequences used for reporter assay of flanking sequence effects. TS1 and TS2 represent miR-223 TS1 (in blue) and TS2 (in green) sequences cloned in psiCheck2.2, respectively. TS1-L and TS2-L represent miR-223 TS1 and TS2 with flanking sequences cloned inpsiCheck2.2, respectively. TS1-L212 represents the miR-223 TS1 sequence cloned in psiCheck2.2 with the TS2 flanking sequences (left in pink and right in cyan). TS2-L121 represents the miR-223 TS2 sequence cloned inpsiCheck2.2 with the TS1 flanking sequences (left in gray and right in red). (b) Reporter assay data showed the target and flanking sequences were not the major factors influencing the effectiveness of TS2. TS2 was actually worse than TS1 when used as short target sequences. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD. (c) Reporter assays of 3′UTR sequence contexts outside of the miR-223 MREs and flanking sequences affecting miR-223 repression. The data show MREs fused with fragment H are more effective than fragment G. miR-223 target sequences in RhoB (TS1 and TS2) and NF-1A (NF1A) were cloned downstream of RhoB 3′UTR fragments G and H (Figure 1b). The data also show that target sites located downstream of fragment H were more effective. Each bar represents the average of at least three independent transfections with duplicate determinations for each construct. Error bars represent the SD.