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. Author manuscript; available in PMC: 2013 May 1.

Published in final edited form as: Nat Immunol. 2012 Oct 7;13(11):1110–1117. doi: 10.1038/ni.2427

(a,b) Immunoassay of 293T cells transfected with the indicated plasmids, followed by denature-immunoprecipitation (Denature-IP) with anti-Flag and immunoblot analysis with anti-ubiquitin (Ub), anti-ubiquitin(K63 specific) or anti-Flag 20 h after transfection. The expression levels of USP25, USP25(C178S) or Act1 were analyzed by immunoblot with anti-Myc or anti-HA. (c) Immunoassay of lysates of wild-type and Usp25^-/- MEFs treated with IL17 (200 ng/ml) for the indicated time points, followed by denature-IP with anti-TRAF5 or anti-TRAF6 and immunoblot with anti-ubiquitin, anti-TRAF5 or anti-TRAF6. (d) Immunoassay of lysates of Usp25^-/-MEFs reconstituted with empty vector (Vector), USP25 (WT) or USP25(C178S) (CS) treated with IL17 (200 ng/ml) for 30 min, followed by denature-IP with anti-TRAF5 or anti-TRAF6 and immunoblot with anti-ubiquitin, anti-TRAF5 or anti-TRAF6. The expression level of reconstituted proteins were analyzed by immunoblot with anti-USP25. Data are representative of three independent experiments.

(a,b) Immunoassay of 293T cells transfected with the indicated plasmids, followed by denature-immunoprecipitation (Denature-IP) with anti-Flag and immunoblot analysis with anti-ubiquitin (Ub), anti-ubiquitin(K63 specific) or anti-Flag 20 h after transfection. The expression levels of USP25, USP25(C178S) or Act1 were analyzed by immunoblot with anti-Myc or anti-HA. (c) Immunoassay of lysates of wild-type and Usp25^-/- MEFs treated with IL17 (200 ng/ml) for the indicated time points, followed by denature-IP with anti-TRAF5 or anti-TRAF6 and immunoblot with anti-ubiquitin, anti-TRAF5 or anti-TRAF6. (d) Immunoassay of lysates of Usp25^-/-MEFs reconstituted with empty vector (Vector), USP25 (WT) or USP25(C178S) (CS) treated with IL17 (200 ng/ml) for 30 min, followed by denature-IP with anti-TRAF5 or anti-TRAF6 and immunoblot with anti-ubiquitin, anti-TRAF5 or anti-TRAF6. The expression level of reconstituted proteins were analyzed by immunoblot with anti-USP25. Data are representative of three independent experiments.