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

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — TRAF5 promotes the ubiquitination of RORγt. A, HEK293T cells were transfected with Myc-RORγt, His-ubiquitin (His-ubi), FLAG-TRAF5, and FLAG-TRAF5ΔN. IB, immunoblot. B and C, His-ubiquitin and its mutants Lys-48 only, Lys-63 only, K48R, and K63R were co-transfected with Myc-RORγt and FLAG-TRAF5 to detect the ubiquitination type for TRAF5 against RORγt. D, human Th17 cells were stimulated with anti-CD3 and anti-CD28 antibodies overnight in fresh medium and then stimulated with different cytokines for 8 h. The cells were treated with MG132 (20 μm) for 4 h before being harvested, and immunoprecipitation (IP) was carried out with anti-ubiquitin antibody. Data represent at least three independent experiments.