FIGURE 3.

IL-17 mRNA is a direct target of HuR. Naive CD4⁺ T cells were isolated from spleen of WT mice and stimulated as described in Fig. 1 in the presence of Th17 cell–polarizing cytokines for 5 d. (A) IP of RNP complexes assayed for detection of IL-17 mRNA in Th17 cell cytoplasmic extracts immunoprecipitated with anti-HuR or isotype-matched Ab. Real-time PCR showed IL-17 mRNA enrichment in the anti-HuR IP samples compared with isotype-matched IgG1. IFN-γ mRNA was hardly detected in any IP samples. (B) Biotin pulldown was used to determine whether HuR directly binds to 3′ UTR of IL-17 mRNA. HuR protein could be detected by Western blotting in biotinylated transcripts spanning IL-17 3′ UTR, but not in IL-17 mRNA ORF biotinylated transcripts. GAPDH biotinylated transcripts were unreactive. Error bars represent ± SEM. Data in (A) were derived from at least three independent experiments. Data in (B) were derived from two independent experiments. **p < 0.001.