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. 2007 Aug 16;26(17):3910–3922. doi: 10.1038/sj.emboj.7601823

Figure 7.

Figure 7

TAX1BP1 promotes the deubiquitination of TRAF6 in an A20-dependent manner. (A) TAX1BP1m/m and TAX1BP1+/m MEFs were treated with IL-1 (20 ng/ml) for the indicated times. Cells were lysed and TRAF6 was immunoprecipitated with anti-TRAF6 followed by immunoblotting with anti-ubiquitin or anti-TRAF6. (B) TAX1BP1m/m and TAX1BP1+/m MEFs were treated with LPS (1 μg/ml) and TRAF6 ubiquitination was examined as described in panel A. (C) 293T cells were transfected with Flag-TRAF6 (1 μg) and HA-Ub (0.5 μg) together with Flag-TAX1BP1 (0.25, 0.5 and 1 μg), Flag-TAX1BP1 1–204 (0.25, 0.5 and 1 μg), Flag-A20 (0.1, 0.25 and 0.5 μg) or Flag-A20 C103A (0.1, 0.25 and 0.5 μg). After 36 h, cells were lysed and immunoprecipitated with anti-TRAF6 followed by immunoblotting with anti-HA. Lysates were examined for TRAF6, TAX1BP1 and A20 expression by immunoblotting with anti-Flag. (D) 293T cells were transfected with vector or Flag-A20 (1 μg) together with pSuper (2 μg) or pSuper A20 nos. 1 or 2 siRNAs (2 μg). After 36 h, cells were lysed and subjected to immunoblotting with anti-Flag. (E) 293T cells were transfected with Flag-TRAF6, HA-Ub and Flag-TAX1BP1 as above together with 2 μg of pSuper or pSuper A20 siRNAs. The IPs and immunoblotting were performed as in panel C. Lysates were examined for TRAF6 and TAX1BP1 expression by immunoblotting with anti-Flag. (F) TAX1BP1 promotes an interaction between A20 and TRAF6. 293T cells were transfected with Flag-A20 (1 μg) and Flag-TRAF6 (1 μg) together with pCAGGS-TAX1BP1 (0, 0.25, 0.5 and 1 μg). After 36 h, cells were lysed and TRAF6 was immunoprecipitated followed by immunoblotting with anti-Flag. Lysates were examined for A20, TRAF6 and TAX1BP1 expression.