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. 2015 Aug 6;6(30):29129–29142. doi: 10.18632/oncotarget.4895

Figure 2. TRAF6 is a direct target of miR-146b-5p.

Figure 2

A. Four miR-146b-5p binding sites in TRAF6 3′-UTR predicted with TargetScan. B. Wild (TRAF6-3′-UTR-WT) and mutant (TRAF6-3′-UTR-MT1 and TRAF6-3′-UTR-MT2) TRAF6 3′-UTRs carried in recombinant luciferase mRNAs transcribed by p-WT, p-MT1 and p-MT2. The binding site 1 and 2 or 3 and 4 were deleted from TRAF6-3′-UTR-MT1 or TRAF6-3′-UTR-MT2. C–E. Luciferase reporter assays in U87MG (C), SNB19 (D) and U251 (E) cells transfected with p-WT, p-MT1 or p-MT2 (Mock), and cotransfected with p-WT, p-MT1 or p-MT2 and scrambled sequence (negative control) or miR-146b-5p mimics. F and G. qRT-PCR analyses of miR-146b-5p mimics-transfecting efficiency (F) and TRAF6 mRNA expression (G) in the cells as indicated. Their relative expression levels were normalized against U6 or GAPDH. The ratios of miR-146b-5p/U6 and TRAF6/GAPDH in untransfected cells (Mock) were arbitrarily set to 1.0. H and I. Western blot analysis of TRAF6 in the cells as indicated. The relative expression level of TRAF6 was normalized against β-actin. All experiments were performed at least in triplicate and the data in C - H are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.