Vif N-terminal amino acids are responsible for Cul5 interaction, in vitro . Vif wild-type and single alanine mutants were co-expressed with N-terminal Cul5, N-terminal 6X-His-CBF-β (residues 1–140), and Elo B/C. Next, the complex was pulled down using nickel affinity purification. A) While Vif wild-type and H28A mutant pull down Cul5 efficiently, H27A, M29A and Y30A mutants are unable to bind Cul5 efficiently. B) Quantitative measurement of the Cul5 band intensity was performed indicating the relative amount of Cul5 bound to Vif wild-type and mutant protein complexes. FL – full length, N-terminal – amino-terminus, 6X-His – 6X histidine tag.