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

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© 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — Co-overexpression of TAK1/TAB1 induces Lys⁶³-linked TAK1 polyubiquitination. A, co-overexpression of TAK1/TAB1 induces TAK1 polyubiquitination. Expression vectors encoding FLAG-TAK1 and HA-ubiquitin were co-transfected into HEK-293T cells with control vector and expression vectors encoding TAB1, respectively. FLAG-TAK1 proteins in the transfected cells were immunoprecipitated (IP) with anti-FLAG antibodies (or anti-HA antibodies) and immunoblotted (IB) with anti-HA antibodies (or anti-FLAG antibodies) to detect the presence of ubiquitinated FLAG-TAK1. B, co-overexpression of TAK1/TAB1 induces Lys⁶³-linked TAK1 polyubiquitination. Expression vectors encoding TAK1-V5-His and TAB1 were co-transfected into HEK-293T cells with control vector and expression vectors encoding HA-ubiquitin wild type, Lys⁶³-only, and Lys⁴⁸-only, respectively. TAK1-V5-His proteins in the transfected cells were immunoprecipitated with anti-V5 antibodies and immunoblotted with anti-HA antibodies to detect the presence of ubiquitinated TAK1-V5-His.