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. Author manuscript; available in PMC: 2022 Jul 19.
Published in final edited form as: Biochemistry. 2019 Jun 18;58(26):2906–2920. doi: 10.1021/acs.biochem.8b01316

Figure 6.

Figure 6.

The 9SG mutation abolishes the ability of NEMO to function in signal-induced activation of IκB kinase. (A) The indicated FLAG-tagged NEMO constructs were transfected into 293T cells, and extracts were immunoprecipitated (IP) with anti-FLAG beads. Immunoprecipitates were then analyzed by anti-FLAG (bottom) and anti-IKKβ (top) Western blotting. In the Input lanes, 4% of the extract used in the immunoprecipitations was analyzed by Western blotting. (B) Whole cell extracts from wild-type and NEMO-knockout 293T cells were analyzed by anti-NEMO Western blotting (top) or Ponceau staining for total protein (bottom). (C) 293T NEMO knockout cells were transfected with plasmids for the expression of 7XAla- or 9SG-NEMO. Cells that were either untreated (−) or treated with TNFα (+) were analyzed by Western blotting for phospho-IκBα or NEMO or by Ponceau staining. (D) Mouse NEMO knockout fibroblasts were stably transduced with retroviral vectors for the indicated NEMO proteins. Stable cell lines were then untreated (−) or treated (+) with the indicated compounds. Extracts were analyzed by Western blotting for the indicated proteins.