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. Author manuscript; available in PMC: 2013 Mar 23.
Published in final edited form as: Immunity. 2008 Mar;28(3):381–390. doi: 10.1016/j.immuni.2008.02.002

Figure 2. A20 Is Required for Restricting MDP-Induced Signaling Activity.

Figure 2

(A) Immunoblotting analyses of phospho-IκBα, IκBα, A20, NOD2, RIP2, and actin protein expression in Tnfaip3−/− and Tnfaip3+/+ BMDMs after MDP stimulation. Tnfaip3−/− and Tnfaip3+/+ BMDMs were stimulated with 10 μg/ml of MDP, and cell lysates were harvested at the indicated time points for immunoblotting analysis. Note increased phospho-IκBα protein expression in Tnfaip3−/− cells at later time points, indicating prolonged NFκB-signaling activity. Note similar amounts of IκBα, NOD2, and RIP2 in Tnfaip3+/+ and Tnfaip3−/− cells after MDP stimulation. Note constant A20 protein expression in Tnfaip3+/+ cells (and not in Tnfaip3−/− cells) after MDP stimulation. Actin protein expression is shown as a control.

(B) IKK kinase assay of Tnfaip3−/− and Tnfaip3+/+ BMDMs after MDP stimulation. Tnfaip3−/− and Tnfaip3+/+ BMDMs were stimulated with MDP, and cell ly-sates were harvested at the indicated time points, immunoprecipitated with anti-IKKγ antibody, incubated with GST-IκBα substrate, and analyzed by immunoblotting for phospho-GST-IκBα levels. Note increased phospho-GST-IκBα expression in Tnfaip3−/− BMDMs indicating prolonged IKK kinase activity. IKKβ protein expression in the immunoprecipitates are shown as a control.