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. Author manuscript; available in PMC: 2012 Jun 1.
Published in final edited form as: Nat Immunol. 2011 Nov 6;12(12):1176–1183. doi: 10.1038/ni.2157

Figure 7.

Figure 7

Ligase activity of Itch, DUB activity of Cyld, and Cyld-Itch interaction is necessary for termination of TNF induced Tak1 activation. (a) Itch−/− MEFs were reconstituted with either V5-Itch(WT) or V5-Itch(C830A) mutant using a lentiviral transduction system. The cells lysed at indicated time points post TNF stimulation. The lysate was immunoprecipitated using against TRAF2 and immunoblotted with antibody against p-Tak1. The same membrane was reprobed with antibody against Tak1. The cell lysate from WT MEFs stimulated with TNF was used as control. (b) Itch−/− MEFs transduced with V5-Itch(WT) or V5-Itch(C830A) mutant as in (a) were stimulated with TNF and expression of IL-6 was analyzed by ELISA in the culture supernatant. (c) Cyld−/− MEFs were reconstituted with V5-Cyld(WT), V5-Cyld(C601A) or V5-Cyld(Y485A) mutant. The cells lysed at indicated time points post TNF stimulation. The lysate was immunoprecipitated using antibody against TRAF2 and immunoblotted with antibody against p-Tak1. The same membrane was reprobed with antibody against Tak1. (d) Cyld−/− MEFs transduced with V5-Cyld(WT), V5-Cyld(Y485A) or V5-Cyld(C601A) mutant as in (c) stimulated with TNF and expression of IL-6 was analyzed by ELISA in the culture supernatant. Data are representative of three (b, d) and two (a, c) experiments. (b,d mean and s.d. of triplicate wells).