Fig 4. Tax-induced generation of IKK complex-associated K63/M1-linked hybrid chains is required for IKK activation.

(A) Jurkat cytosolic extracts were incubated with recombinant His₆-Tax and ATP (2 mM), and the reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody. The immunoprecipitates were left untreated or were treated with recombinant Otubain-1, AMSH, OTULIN or USP2 and analyzed by immunoblotting with anti-Ub (upper) and anti-M1 chain-specific (lower) antibodies. (B) Jurkat cytosolic extracts were incubated with or without recombinant His₆-Tax and ATP (2 mM), and the reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody. The immunoprecipitates were analyzed via the ubiquitin-AQUA method. The results are given as the mean ± SD (n = 3). (C) Cell-free reactions were performed in the presence of ubiquitin mutants or HA-ubiquitin (50 μM). The reaction mixtures were subjected to immunoprecipitation with an anti-NEMO antibody, followed by immunoblotting with an anti-Ub antibody. (D) Cell-free reactions were performed in the presence of various DUBs. The depicted results are representative of three independent experiments.