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. 2015 Oct 9;290(48):29086–29094. doi: 10.1074/jbc.M115.664573

FIGURE 2.

FIGURE 2.

TRAF5 interacts with and stabilizes RORγt. A, TRAF family members and Myc-RORγt were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated (IP) with anti-Myc antibody. IB, immunoblot. B, Myc-RORγt and FLAG-TRAF5 or FLAG-TRAF5ΔN were co-transfected into HEK293T cells. Cell lysates were immunoprecipitated with either anti-Myc or anti-FLAG antibody. C, naïve CD4+ T cells were polarized under Th17 conditions, and then Th17 cells were harvested and lysed with radioimmune precipitation assay buffer. The cells lysate was immunoprecipitated with anti-TRAF5 antibody or a rabbit IgG control. Th17 cells were stimulated by anti-CD3 and anti-CD28 antibodies after resting for 1 day, and cells were collected at 0, 1, 4, and 8. D, HEK293T cells were co-transfected with Myc-RORγt and the indicated doses of FLAG-TRAF5 or its mutant (0, 0.4, 0.8, and 1.2 μg). The statistical curve was determined as shown. E, Myc-RORγt with or without FLAG-TRAF5 or TRAF5ΔN was transfected into HEK293T cells. The half-life of RORγt in cycloheximide (CHX)-treated cells was analyzed as shown. Ctrl, control. Data represent at least three independent experiments.