Human CRL2^ZYG11B interacts with cyclin B1 independently of Cdk1. (A) FLAG-ZYG11B interacts with Venus–cyclin B1 mutant proteins that are incapable of binding Cdk1. FLAG-ZYG11B was coexpressed with wild-type or mutated versions of Venus–cyclin B1 with R202A or R202A R42A substitutions in HEK293T cells that were treated with MG132. The Venus tag was immunoprecipitated and immunoblotted for Venus–cyclin B1, FLAG-ZYG11B, or Cdk1 (left). Similar results were obtained in two independent experiments. (B) Endogenous Cdk1 coprecipitates HA-ZYG11B but not control HA–leucine-rich repeat (LRR) 1 in HEK293T cells treated with MG132. Similar results were obtained in two independent experiments. (C) HA-ZYG11B predominantly interacts with the CBOX1 domain of cyclin B1. HA-ZYG11B was coexpressed with Venus–cyclin B1, Venus-tagged truncations of cyclin B1 (N terminus, CBOX1, or CBOX2), or pEGFP-N1 control vector in HEK293T cells treated with MG132. Note that the truncations were cleaved into smaller isoforms in vivo (the full-length size is marked by an arrow on the right side). The Venus tag was immunoprecipitated and immunoblotted for HA or Venus tags (left panels). The results are representative of five independent experiments.