HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains
Fig 5
Tax induces formation of the macromolecular active IKK complex.
(A) Jurkat cytosolic extracts were incubated with recombinant His6-Tax or His6-MEKK1 together with ATP (2 mM) in the presence or absence of IsoT. (B) Cell-free reactions were performed in the presence of WT viral OTU or its catalytic inactive mutant 1A. (C) Jurkat cytosolic extracts were incubated with recombinant His6-Tax or its mutants together with ATP (2 mM). The reaction mixtures were subjected to immunoprecipitation with an anti-Tax antibody, followed by immunoblotting with an anti-Ub antibody. (D) Cytosolic extracts were prepared from NEMO-deficient MEFs reconstituted with human NEMO or its mutant and were subjected to cell-free analyses. (E) Cytosolic extracts were prepared from NEMO-deficient MEFs reconstituted with mouse NEMO or its mutants and were subjected to cell-free analyses. (F) Cell-free reactions were performed, and the reaction mixtures were subjected to Blue native-PAGE, followed by immunoblotting with anti-NEMO (left) and anti-p-IKKα/β (right) antibodies. The depicted results are representative of three independent experiments.