Cif interacts <i>in vitro</i> with the ubiquitin-like protein NEDD8.

<p>(<b>A</b>) Hexa-histidine tagged wild-type Cif (WT), Cif cysteine 109 mutant Cys-to-Ser (C/S), Cif deleted for amino-acids 70-85 mutant (Δ70-85) or Cif Δ70-85 Cys-to-Ser mutant(Δ70-65 C/S) and NEDD8 or ubiquitin (Ub) proteins were co-expressed in bacteria, and then His-Cif proteins were purified from bacterial lysates using nickel affinity. Whole extracts and eluted fractions were probed with indicated antibodies. (<b>B</b>) Wild-type His-Cif and NEDD8 were co-expressed in bacteria and His-Cif was purified using nickel affinity and gel filtration chromatography. The peak in the chromatograph corresponds to the His-Cif complex. NEDD8 and the corresponding fractions were visualized using SDS-PAGE stained with Coomassie blue. (<b>C</b>) Glutathione sepharose matrix was mixed with 100 µg of GST or GST-Cif Cys-to-Ser mutant purified proteins and then incubated with a lysate of HEK293T cells expressing a FLAG-NEDD8 construct. 100 µg or 250 µg of cells extract were used to obtain a GST constructs/cell extract ratio of 1∶1 and 1∶2.5 respectively. Cells extract (input) and eluted fractions were immunoblotted with anti-FLAG antibodies. FLAG-NEDD8 fusion protein is indicated with an arrow.