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. 2015 Aug 4;5:12738. doi: 10.1038/srep12738

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Copyright © 2015, Macmillan Publishers Limited

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(a) USP18 blocks K63-linked polyubiquitination of NEMO. Lysates of 293T cells transfected with plasmids were immunoprecipitated with anti-Flag and immunoblotted with anti-HA. (b) Knockdown of USP18 increased K63-linked ubiquitination of NEMO. Immunoprecipitation and immunoblot analyses were performed after co-transfection of Flag-NEMO, HA-K63 ubiquitin, along with USP18 siRNA or scrambled siRNA in 293T cells. (c) Lysates of 293T cells transfected with plasmids were immunoprecipitated anti-Flag, then boiled proteins in presence of 1% SDS, followed by a second immunoprecipitation with anti-Flag. (d) USP18 mutations were able to inhibit K63 linked ubiquitination of NEMO. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with Flag-NEMO, HA-K63 ubiquitin, and Myc-USP18 (WT), Myc-USP18 (C64S), or Myc-USP18 (C64S H318A). (e) The inhibition of USP18 in NF-κB is independent of its protease activity. 293T cells were transfected with an NF-κB luciferase reporter, together with vector for MyD88, TAK1+TAB1, IKKβ, along with wild type USP18 or its mutants. The expression of the IKKβ and USP18 mutants were shown by immunoblot. (f) The domain structure of NEMO. (g) UBAN domain of NEMO is essential for its interaction with USP18. The Flag-tagged different deletion constructs of NEMO were co-transfected with Myc-USP18 in 293T cells and immunoprecipitation and immunoblot were performed. (h) Immunoprecipitation and immunoblot analysis of 293T cells transfected with various combinations (above lanes) of plasmid for Myc-tagged USP18 and HA-tagged K63-linked ubiquitin together with plasmid for Flag-tagged NEMO mutations. (i) USP18 cannot inhibit NF-κB activation in NEMO knockout cells which transfected with NEMO (K325/326R). NEMO knockout cells were transfected with NEMO (WT), NEMO (K285/309R) mutant or NEMO (K325/326R) mutant and other indicated plasmids. Data in i are presented as the means ± SD of three independent experiments. ***P < 0.001 versus cells transfected with control vector or USP18 (Student’s t-test).