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. 2014 Jul 28;289(37):25546–25555. doi: 10.1074/jbc.M114.565291

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© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — USP17 deubiquitinates Lys-48-linked RORγt. A, 293T cells were transfected with Myc-tagged USP17 and FLAG-tagged ubiquitin (Ubi). Forty-eight hours later, the cell lysates were harvested, and immunoprecipitation (IP) was performed with anti-Myc antibodies plus protein A/G beads. IB, immunoblot. B, 293T cells were transfected with FLAG-tagged RORγt, Myc-tagged USP17, Myc-tagged USP17C89S (CS), and His-tagged ubiquitin. The ubiquitin pulldown assay for FLAG-tagged RORγt was performed as described under “Experimental Procedures.” C, the His-tagged ubiquitin mutants Lys-48only (K48only) and Lys-63only (K63only) were cotransfected with FLAG-tagged RORγt and Myc-tagged USP17 to detect the deubiquitination site for USP17 against RORγt. The ubiquitin pulldown assay was performed as described under “Experimental Procedures.” Data are representative of more than three independent experiments.