KLHL21 binds and mediates ubiquitination of Aurora B. (A) HeLa cells expressing photoactivatable GFP (PA-GFP) or GFP–Aurora B were treated with 100 ng/ml nocodazole for 16 h followed by mitotic shake-off. Extracts were prepared and immunoprecipitated with anti-GFP antibodies. Inputs and eluates (GFP IP) were analyzed by immunoblotting. (B) HeLa cells expressing GFP–Aurora B were transfected with an empty vector or vector containing HA-BTBD1, -KLHL21, or –KLHL21-DLQ. Extracts were immunoprecipitated with anti-GFP, and inputs and eluates (GFP IP) were analyzed by immunoblotting. (C and D) Recombinant GST and GST-6-Kelch–KLHL9 and –KLHL21 were incubated with recombinant Aurora B (C) or survivin (D). Mixtures were bound to glutathione Sepharose beads, and inputs and eluates (GST pd) were analyzed by immunoblotting. (E and F) Recombinant Aurora B was added to in vitro ubiquitination reactions containing GST-Cul3–Rbx1 complexes and UbcH5a. Reactions were incubated for 1 h with MBP or MBP-KLHL21, -KLHL9, or -KLHL13 and analyzed by immunoblotting. In the presence of ATP, Aurora B migrated slower because of autophosphorylation (asterisks). In F, methyl-ubiquitin was added instead of ubiquitin. Note that Aurora B was mainly monoubiquitinated (arrows) under these conditions, but multiple sites were used with lower efficiency.