Figure 2

Binding of miR-133b to NUP214 is specific. (a) and (c) Cartoons showing the two NUP214 3′UTR constructs used for luciferase assay. (a) represents wild type NUP214 3′UTR downstream of luciferase gene while (c) shows NUP214 3′UTR where the target site for miR-133b is mutated. (b) miR-133b decreases luciferase activity in presence of NUP214 3′UTR. UPCI:SCC084 cells were transiently co-transfected with 0.5 μg pSB-NUP214/3′UTRLuc and 0, 0.25, 0.5, 1 or 2 μg of pSB-miR-133b. Protein lysates were prepared for luciferase assay. (d) Mutant NUP214 3′UTR prevents inhibition by miR-133b. UPCI:SCC084 cells were co-transfected with either 0.5 μg pSB-NUP214/3′UTRLuc, or pSB-NUP214/3′UTRMutLuc along-with 0 or 1 μg pSB-miR-133b. Protein lysates were prepared for luciferase assay. (e) Blocking miR-133b prevents NUP214 repression. UPCI:SCC084 cells were transiently transfected with only pSB-NUP214/3′UTRLuc (0.5 μg), or with pSB-NUP214/3′UTRLuc (0.5 μg) and pSB-miR-133b (1 μg), or with pSB-NUP214/3′UTRLuc (0.5 μg), pSB-miR-133b (1 μg) and anti-miR-133b (20 nM). Protein lysates were prepared for luciferase assay. For (b), (d) and (e), data represent three independent experiments and are shown as average ± S.D.