Fig. 7.

A20-dependent degradation of endogenous TRAF2. (A) Impaired degradation of endogenous TRAF2 in A20 deficient MEF cells. Wild type (A20+/+) or A20 deficient (A20−/−) MEF cells were treated with TNFα (40 ng/ml) and actinomysin D (1μg/ml) for the indicated time points. Cell extracts were analyzed by immunoblotting (IB). Graph shows the quantification of four independent experiments. Error bars represent S.D. (n=4). (B) Inhibition of TRAF2 degradation by a lysosomal inhibitor. HeLa cells were treated with TNFα and actinomysin D for the indicated time periods and analyzed as in (A). Where indicated, cells were pretreated with NH₄Cl for 1 h. Numbers indicate the relative level of TRAF2. Shown is a gel representing 3 independent experiments. (C) TWEAK induces the loss of endogenous TRAF2 by a lysosome dependent mechanism. HeLa cells were treated with the indicated protein/chemical (TWEAK, 100 ng/ml; chloroquine, 80 μM) for 6 h. Cell extracts were analyzed by immunoblotting. Asterisk indicates a non-specific band. (D) The specificity of the TRAF2 antibody as demonstrated by immunoblotting analyses using whole cell extract from the indicated cell lines. (E) TWEAK-mediated TRAF2 turn over requires A20. Immortalized wild type (+/+) and A20 knock-out cells were treated for 6 h as indicated. Whole cell extract was subjected to immunoblotting analysis.