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. 2018 Jan 3;4(1):e1701393. doi: 10.1126/sciadv.1701393

Fig. 2. Confirmation of the activity of the selected E4B U-box mutants in mediating UB transfer with xUbcH5b.

Fig. 2

(A) wt UB could be efficiently transferred through the xUba1(UFD)-xUbcH5b pair to the KB2 and KB12 mutants displayed on the phage surface. wt UB transfer through the wt Uba1-UbcH5b pair to wt U-box of E4B on phage surface was set up as a control. IB, immunoblotting. (B) U-box mutants KB2, KB7, KB9, KB11, and KB12 from phage selection were expressed as fusions with a flag tag, and their autoubiquitination with wt UB though the xUba1(UFD)-xUbcH5b pair was analyzed by Western blotting. (C) Similar to (B), U-box mutants were subjected to xUB transfer through the xUba1-xUbcH5b pair. In both cases, efficient modification of the U-box by wt UB and xUB was observed. Reactions were stopped at the stage of monoubiquitination to compare the reaction efficiency among various U-box mutants.