An intact UIM motif is necessary for UBXN7 interaction with neddylated-CUL2. (A) Schematic representation of human UBXN7 highlighting its various domains. (B-D) Wild-type or mutant Flag-UBXN7 was immunoprecipitated from HeLa cells. The indicated proteins were detected using specific antibodies in the immunoprecipitates (left) and the input cell extracts (right). (B) Either UBA- or UIM-deletion caused a reduction in ubiquitin-binding to UBXN7. UBX-deletion or a point mutation in this domain (P459G) abolished p97-binding and severely impaired the interaction with ubiquitylated proteins (left panel). (C) UIM-deletion, but not other mutations, caused a strong reduction in CUL2-binding to UBXN7 (left panel) and abolished the CUL2 up-shift caused by UBXN7 over-expression (right panel). (D) Point mutations within the UIM motif at Ser297 caused a defect in CUL2-binding to UBXN7 similar to UIM-deletion. p97-binding was not affected by these mutations (left panel).