Cif interacts with NEDD8-conjugated cullins.

<p>HeLa cells were infected 3 h with EPEC<i>Δcif</i> carrying the empty vector (pBRSK) or plasmids expressing His-tagged <i>E. coli</i> Cif cysteine mutant (C/A), 70-85 deleted mutant (Δ70-85) or wild-type (WT) and further incubated 3 h with antibiotics. Cell extracts were immunoprecipitated with anti-His antibodies. (<b>A</b>) Total (tot), non-retained (nr) and immunoprecipitated (ip) protein fractions were probed with anti-NEDD8 antibodies. Arrows indicate neddylated cullins (top) and monomeric NEDD8 (bottom). Molecular weights in kDa are indicated. (<b>B</b>) Total and immunoprecipitated protein fractions were probed with anti-Cif, anti-cullins (CUL1 to CUL4B), anti-RBX1, anti-CAND1, anti-Skp1, anti-Skp2 and anti-actin antibodies. (<b>C</b>) High loads of immunoprecipitated fractions were probed with anti-cullins (CUL2 and CUL4B) antibodies.