UBXN7 exclusively interacts with neddylated cullins in cell extracts. (A) UBXN7 preferentially interacts with slower-migrating, neddylated CUL2. Input cell extracts and the supernatants after Flag-UBXN7 immunoprecipitation were compared. (B) Flag-UBXN7 over-expression causes an up-shift of CUL2 to its neddylated form. This effect was abolished when the cells were grown in the presence of the NEDD8-E1 inhibitor MLN4924 for two hours. (C) Alignment of human CUL2 with the yeast cullin Cdc53. The conserved neddylation site (K689) and a more C-terminal Lys residue involved in the interaction with Dcn1 (K719) are highlighted in red. (D) Neddylation-defective CUL2 variants are similarly defective in interacting with endogenous UBXN7. Wild-type or mutant Flag-CUL2 was immunoprecipitated from HeLa cells treated or not with 10 μM MG132 for two hours. (E) MLN4924 treatment abolishes UBXN7 interaction with several endogenous cullins. (F) MLN4924 treatment has no effect on UBXN7 interaction with ubiquitylated-proteins or with p97. (E, F) Flag-UBXN7 was immunoprecipitated from HeLa cells treated with MG132, MLN4924, or a combination of the two. The indicated proteins were detected using specific antibodies.