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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Science. 2010 Feb 26;327(5969):1135–1139. doi: 10.1126/science.1182364

Fig. 4.

Fig. 4

Requirement of A20 zinc finger 4 for TAX1BP1 binding and degradation of Ubc13 and UbcH5c. (A) Schematic of A20 deletion mutants. (B) Requirement of A20 ZnF4 for the degradation of Ubc13. A20−/− MEFs were transiently transfected with the indicated mutants. After 36 hours, cells were treated with TNF-α for the indicated times. Lysates were subjected to immunoblotting with antibodies to Ubc13, IκBα, β-actin, or Flag. (C) A20 DUB and ZnF4 mutants do not interact with Ubc13. A20−/− MEFs were transiently transfected with Flag-A20, Flag-A20 (C103A), and Flag-A20 ZnF4 mutant (C624A and C627A). After 36 hours, cells were stimulated with LPS for the indicated times, and proteins from lysates were immunoprecipitated with Ubc13 antibody and detected by immunoblotting with antibodies to A20 or Ubc13. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα, and β-actin. (D) A20 ZnF4 mutant does not interact with Ubc13. A20−/− MEFs were transiently transfected with Flag-A20 and Flag-A20 ZnF4 mutant (C624A and C627A). After 36 hours, cells were stimulated with TNF-α for 60 min, and proteins from lysates were immunoprecipitated with Ubc13 antibody and detected by immunoblotting with antibodies to Flag or Ubc13. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. (E) A20 DUB and ZnF4 mutants do not interact with UbcH5c. A20−/− MEFs were transiently transfected with plasmids as described in (C). After 36 hours, cells were stimulated with IL-1 for the indicated times, and proteins from lysates were immunoprecipitated with UbcH5c antibody and detected by immunoblotting with antibodies to A20 or UbcH5c. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. (F) Differential requirement for the A20 OTU domain and C-terminal zinc fingers in binding to TAX1BP1, Ubc13, and UbcH5c. A20−/− MEFs were transiently transfected with empty vector, Flag-A20, Flag-A20 (547-775) or Flag-A20Δ547-699. After 36 hours, cells were treated with IL-1 for either 30 or 60 min. Proteins from lysates were immunoprecipitated with antibody to Flag, followed by immunoblotting with antibodies to TAX1BP1, UbcH5c, or Ubc13. Lysates were subjected to immunoblotting with antibodies to IκBα, Flag, and β-actin. (G) A20 ZnF4 mutant does not interact with TAX1BP1. A20−/− MEFs were transiently transfected with plasmids as described in (C). After 36 hours, cells were stimulated with LPS for the indicated times, and proteins from lysates were immunoprecipitated with TAX1BP1 antibody and detected by immunoblotting with antibodies to A20 and TAX1BP1. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. The results shown are representative of three independent experiments.