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. 2014 Feb 4;22(2):337–344. doi: 10.1016/j.str.2013.12.004

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© 2014 The Authors

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Additional Residues of Ube2T Are Required for Binding the RING Domain of FANCL

(A) Size-exclusion chromatogram profiles of wild-type (WT) or mutant Ube2T (blue) dotted line and WT RING domains (green dashed line) overlain with profiles from binding experiments in which WT RING domain has been incubated with WT or mutant Ube2T (pink line) and subjected to size-exclusion chromatography. Binding was assessed by complex formation indicated by a peak shift to the left.

(B) An anti-HA-Ub western blot of in vitro monoubiquitination assays to assess monoubiquitination of FLAG-FANCD2 by FANCL in collaboration with different WT Ube2T and Ube2T mutants (Ube2TArg99Ser/Ser101Arg, Ube2TSer5Arg, and Ube2TArg60Glu). Lanes 2, 4, and 6 show the monoubiquitination of FLAG-FANCD2 when WT Ube2T, Ube2T Arg99Ser/Ser101Arg, and Ube2TSer5Arg are paired with FANCL. Monoubiquitination is not observed for with the Ube2TArg60Glu mutant is used (lane 8).