HHR23A mutations that disrupt the UBL-UBA interaction enhance ubiquitin-binding. (A) GST-HHR23A, untagged mutant HHR23A, free polyubiquitin chains, and glutathione-sepharose beads were mixed together. After the resin was washed, the bound proteins were eluted and resolved by SDS-PAGE and Western blotting performed with an antibody against polyubiquitin. While all the bands are conjugates of free ubiquitin, the arrow indicates that which corresponds to tetraubiquitin. (B) The band corresponding to tetraubiquitin was quantified with a Biorad Fluor-S Max phosphoimager and data from a representative experiment are presented here.