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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-b) USP18 interacts with TAB1 and TAK1. Co-immunoprecipitation and immunoblot analysis of 293T cells transfected with various combinations (above lanes) of plasmids for Flag-TAB1, HA-TAK1, and Myc-USP18. (c) USP18 interacts with IKKα/β and NEMO. 293T cells were transfected with HA-IKKα, HA-IKKβ, HA-NEMO, and Myc-USP18. HA-tagged IKK protein was immunoprecipitated with anti-HA beads and blotted with anti-Myc. (d) 293T cells were transfected with HA-IKKα, HA-IKKβ, HA-NEMO, and Flag-USP18. Flag-tagged USP18 was immunoprecipitated with anti-Flag beads, and blotted with HA. (e) The domain structure of IKKβ. Numbers in parentheses indicate amino acid position in construct. KD, kinase domain; LZ, leucine zipper; HLH, helix-loop-helix. (f) The interaction between Myc-USP18 and Flag-IKKβ domain. 293T cells were transfected with Myc-USP18 and Flag-IKKβ or various Flag-IKKβ domains. Whole cell extracts were immunoprecipitated with anti-Flag beads, and blotted with anti-Myc. (g) NEMO is essential for the interaction between USP18 and IKK. Wild type (WT) 293T cells and NEMO knockout (KO) 293T cells were transfected with Flag-USP18 and HA-TAK1, HA-IKKα or HA-IKKβ. Whole cell extracts were immunoprecipitated with anti-HA beads, and blotted with anti-Flag. (h) Immunoassay of extracts of THP-1 derived macrophages treated for various times (above lanes) with LPS, followed by immunoprecipitation with anti-USP18 and immunoblot analysis (antibodies, left margin).