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. 2015 Jul 20;5:12300. doi: 10.1038/srep12300

Figure 5. Polyubiquitination of TAK1 at Lys562 is required for the activation of TAK1 and MAPK, but not IKK.

Figure 5

A, mutation in Lys562 residue of TAK1 in IKK, in response to LPS. TAK1-silenced RAW264.7 cells were reconstituted with Flag-tagged TAK1 wild-type (WT) or K(562,563)R mutant. The cells were incubated with LPS for the indicated times, lysed and phosphorylated proteins were analyzed with immunoblotting (n = 3). B and C, expression of the TAK1 K(562,563)R mutant lead to declined TLR4-mediated AP-1 reporter activity, but had no effect on NF-κB reporter activity. Flag-tagged TAK1 wild-type (WT) or K(562,563)R mutant, Renilla-Luc plasmid and either AP-1 or NF-κB reporter were co-expressed in HEK293T cells with or without TLR4-MD2 for 24 h. Relative luciferase activity was measured and normalized with the Renilla activity. Results are averages ± SD of three separate experiments (*, significant difference).