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. 2010 Nov 1;31(1):118–126. doi: 10.1128/MCB.00818-10

FIG. 2.

FIG. 2.

Mapping of the amino acid residues required for Ub binding. (A) Comparison of the UMI, UIM, and MIU motifs, where residues involved in the binding to Ub are in bold; underlined sequences were drawn as projected helices using the Helical Wheel Projections software tool (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). (B) The point mutants were generated by introduction of single (A151G, L149A, or L150A) or double (L149A and L150A) amino acid substitutions addressing the sequence 134 to 166 and tested by in vitro pulldown assay as in Fig. 1D. (C) The indicated RNF168 point mutations were inserted within the sequence encompassing amino acids 56 to 571 and analyzed by in vitro pulldown assay using K48 (left panel) and K63 (right panel) poly-Ub chains. Anti-Ub and anti-GST immunoblots are shown.