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. 2021 Aug 4;2021:9937243. doi: 10.1155/2021/9937243

Figure 4.

Figure 4

IL-6Rα is a direct target of HuR. Naïve CD4+ T cells were stimulated under Th17 cell-polarizing cytokines for 5 days. (a) The expression of IL-6Rα and gp130 mRNAs in WT and HuR KO Th17 cells for 4 days of culture was measured by RT-qPCR. (b) The data of western blot assay confirmed that HuR protein was deleted in HuR KO Th17 cells. (c, d) The result of flow cytometry assay showed that IL-6Rα protein level decreased in KO Th17 cells compared with WT Th17 cells. (e) Summary of IL-6Rα-positive cells (percentage) from three independent flow cytometric assay experiments is shown (mean ± SEM). (f) RIP assay showed that HuR directly bound to IL-6Rα, but not Jak1 and Jak2 mRNA. One RIP assay result is shown here. The repeated RIP assay got the same tendency of results but data are not shown here. (g) IL-6Rα mRNA half-life was much shorter in HuR KO Th17 cells than in WT Th17 cells, as determined by actinomycin D treatment following RT-qPCR assay. One of the two repeat experiments is shown. (h–j) There was no remarkable difference in Jak1 and Jak2 mRNA and protein abundance between WT and HuR KO Th17 cells. (i) The levels of p-Jak1 decreased in HuR KO Th17 cells at the indicated time, (j) but not p-Jak2. Data in (a), (e), and (h) represent the summary of three independent experiments. Student's t-test was used for statistics analysis. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Data in (b)–(d) represent one of the three independent experiments. Data in (f), (g), (i), and (j) represent one of the two independent experiments.