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. Author manuscript; available in PMC: 2017 Aug 15.
Published in final edited form as: J Immunol. 2016 Jul 18;197(4):1148–1158. doi: 10.4049/jimmunol.1600548

Figure 5.

Figure 5

USP15 regulates RORγt activity and Th17 differentiation via enhancing SRC recruitment. (A) IL-17 reporter was transfected into 293T cells together with vector (open bars) or SRC1 expression plasmid (black bars) and indicated plasmids for 24 h. Luciferase activity was then measured. Data represent mean ± SE from three independent experiments with at least two replicas in each experiment. **<0.01. ns stands for not significant. (B) RORγt or K446R was expressed together with SRC1 in the presence or absence of USP15 in 293T cells for 48 h. IP with αRORγt was performed to detect RORγt-SRC1 interactions. Immunoprecipitated SRC1 relative to that immunoprecipitated by wild type RORγt was calculated. (C) RORγt or K446R was expressed with SRC1 in the presence or absence of inactive USP15 C298A mutant in 293T cells for 48 h. IP with αRORγt was performed to detect RORγt-SRC1 interaction. Immunoprecipitated SRC1 relative to that immunoprecipitated by wild type RORγt was calculated. (D–F) Retrovirus expressing GFP with scrambled shRNA (Ctrl) or shRNAs specific for knockdown of USP15 or USP45 was transduced into CD4+ T cells and differentiated under Th17 conditions for three days. Intracellular IL-17 was detected by flow cytometer. E is the summary of three independent experiments. Knockdown of USP15 and USP45 were monitored by IB in F. (G) RNA was prepared from control (open bars), shUSP15-1 (grey bars)- or shUSP15-2 (black bars)-transduced T cells that differentiated under Th17 conditions. qPCR was performed to quantitate expression of the indicated genes. * P < 0.05, ** P<0.01. ns stands for not significant. (H–J) Retrovirus expressing GFP alone (EV) or together with USP15 WT or inactive C298A mutant were transduced into CD4+ T cells and differentiated under Th17 conditions for three days. Intracellular IL-17 was detected by flow cytometry. (I) is the summary of three independent experiments. USP15 and GFP expression was detected by westernblot analysis in J, and expression levels relative to wild type RORγt was calculated. Unless specified, data shown are the representative of at least three independent experiments.