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. 2010 Jul 12;107(30):13444–13449. doi: 10.1073/pnas.0913690107

Fig. 2.

Fig. 2.

Mule ubiquitinates Miz1 in vitro. (A) GST-Xpress-Miz1 was incubated with HA-ubiquitin in the presence or absence of immunoprecipitated Mule (WT or C4341A mutant) in an in vitro ubiquitination assay in the presence or absence of ATP, and E1/E2, as indicated. Ubiquitination of GST-Xpress-Miz1 was analyzed by immunoblotting with anti-Xpress antibody (Left) or anti-HA antibody (Right). (B) In vitro ubiquitination assays with GST-Miz1 and recombinant Mule. (C) Ubiquitination of GST, GST-IκBα(1-54), or GST-Miz1 by recombinant Mule in an in vitro ubiquitination assay. (D) In vitro ubiquitination assays with GST-Miz1 in the presence of ubiquitin K48O or K63O. (E) Ubiquitination catalyzed by GST-TRAF2 in an in vitro ubiquitinaiton assay in the presence of ubiquitin K63O. The polyubiquitin chain formation was analyzed by immunoblotting with antiubiquitin antibody.