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. 2009 Dec 28;285(8):5347–5360. doi: 10.1074/jbc.M109.076976

FIGURE 6.

FIGURE 6.

IKKγ/NEMO binds to polyubiquitinated TAK1. A, recombinant IKKγ/NEMO pulls down polyubiquitinated TAK1. Expression vectors encoding TAB1 and HA-ubiquitin were co-transfected into HEK-293T cells with expression vectors encoding TAK1-V5-His wild type and K158R mutant, respectively. The cell lysates were co-incubated with recombinant GST control and GST-IKKγ/NEMO proteins bound to glutathione-agarose beads for 2 h, respectively. GST control and GST-IKKγ/NEMO proteins bound to glutathione-agarose beads were first precipitated with centrifugation and boiled. Then co-precipitated TAK1-V5-His proteins were immunoprecipitated (IP) with anti-V5 antibodies and immunoblotted (IB) with anti-HA antibodies to detect the presence of polyubiquitinated TAK1-V5-His proteins. B and C, TNFα (B) and IL-1β (C) induce association of IKKγ/NEMO with TAK1 wild type but not K158R mutant proteins. HeLa cells with stable expression of FLAG-TAK1 wild type and K158R mutant were either untreated or treated with TNFα (10 ng/ml) (B) and IL-1β (10 ng/ml) (C) for the times indicated and subsequently lysed. Endogenous IKKγ/NEMO proteins in the cell lysates were immunoprecipitated with anti-NEMO antibodies and immunoblotted with anti-FLAG antibodies to detect the co-immunoprecipitation of FLAG-TAK1.