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

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Copyright © 2015, Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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A, Lys562 or Lys563 residue of TAK1 undergoes non Lys48-linked polyubiquitination. Flag-tagged TAK1 wild-type (WT), K(545,546)R, or K(562,563)R mutants were co-expressed in HEK293T cells with HA-tagged Ub WT, K48R, or K63R mutants, and analyzed for polyubiquitination as described above. B, in response to LPS, Lys562 or Lys563 residue of TAK1 undergoes Lys63-linked polyubiquitination. 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, immunoprecipitated, and detected polyubiquitination with anti-Lys63-linked Ub antibodies. C, Lys562 residue of TAK1 undergoes Lys63-linked polyubiquitination. Flag-tagged TAK1 wild-type (WT), K562R, K563R, or K(562,563)R mutants were co-expressed in HEK293T cells with HA-tagged K48R mutants. Lysates of the cells were analyzed for polyubiquitination. D, TAK1 polyubiquitnation enhanced by blocking TAK1 phosphorylation is located at the Lys562 residue. Flag-tagged TAK1 wild-type (WT), T(184,187)A, K(562,563)R, or T(184,187)A, K(562,563)R mutants were co-expressed in HEK293T cells with HA-tagged Ub WT, K48R, or K63R mutants. The cells were collected and analyzed for polyubiquitnation. E, TAK1 polyubiquitnation enhanced by TAK1 inhibition is located at the Lys562 residue. Flag-tagged TAK1 wild-type (WT), or K(562,563)R mutant was co-expressed in HEK293T cells with HA-tagged Ub K48R mutant. After treatment with TAK1 inhibitor 5Z-7-oxozeanenol for 30 min, the cells were collected and analyzed for polyubiquitnation. F, polyubiquitination at Lys158 residue of TAK1 is independent to Lys562 residue. Flag-tagged TAK1 T(184,187)A, T(184,187)A, K(562,563)R, or T(184,187)A, K(158,562,563)R mutants were co-expressed in HEK293T cells with HA-tagged Ub K48R mutant. The cells were collected and analyzed for polyubiquitnation. The result are represented from at least three independent experiments.