Fig. 2.

AREs within the IL-23 mRNA 3′UTR are essential for the inhibitory effect of TTP. (A) Schematic representation of the luciferase reporter constructs used in this study. Fragments and oligonucleotides derived from the IL-23 mRNA 3′UTR were cloned upstream of the luciferase reporter gene in the psiCHECK2 luciferase expression vector. White circles represent the wild-type (w) pentameric motif, AUUUA, and black circles represent the mutated (m) motif, AGCA. (B, C) TTP overexpression inhibited activity of the luciferase reporter containing the IL-23 3′UTR. (B, C) 293 EBNA (B) and CT26 (C) cells were co-transfected with luciferase reporters containing full-length IL-23 mRNA 3′UTR (IL-23 3′UTR Full) and with either pcDNA/V5-TTP or the empty pcDNA/V5 control vector. Cells were harvested and Renilla luciferase activity was normalized to firefly activity. The luciferase values obtained from cells cotransfected with the luciferase construct and pcDNA6/V5 were set to 1. Results shown represent the mean ± SD of three independent experiments (p < 0.05). (D) Mapping of the region in the IL-23 mRNA 3′UTR that is required for TTP inhibition of luciferase activity. CT26 cells were cotransfected with 0.5 μg of a luciferase reporter construct as described in (A) and either pcDNA6/V5-TTP or the empty vector pcDNA6/V5. After normalization of luciferase activity, the TTP-induced inhibition of luciferase activity observed with each construct was compared with inhibition obtained with the full-length IL-23 mRNA 3′UTR. Results shown represent the mean ± SD of three independent experiments (P < 0.001).