Human CRL2^ZYG11A/B physically interacts with cyclin B1. (A) IP of endogenous cyclin B1 coprecipitates FLAG-ZYG11B in HEK293T cells treated with MG132. Left panels are for cyclin B1 IP and right panels are whole cell lysate. Similar results were obtained in two independent experiments. (B) In vitro ubiquitylation of cyclin B1 by the CRL2^ZYG11B complex. T7-tagged cyclin B1 and FLAG-ZYG11B were coexpressed in HEK293T cells in the presence of MG132. The CRL2^ZYG11B complex was immunoprecipitated with anti-FLAG antibody (1st IP). The immunocomplex (with associated T7–cyclin B1) was used in an in vitro ubiquitylation reaction with HA-tagged ubiquitin (Ub). T7–cyclin B1 was subsequently immunoprecipitated in the presence of 0.1% SDS (2nd IP), followed by immunoblotting for HA-ubiquitin and T7–cyclin B1. Similar results were obtained in two independent experiments. (C) Western blot showing specificity of anti-ZYG11A and anti-ZYG11B antibodies on whole cell lysate from HEK293T cells overexpressing FLAG-CDT2, FLAG-ZYG11A, or FLAG-ZYG11B. Similar results were obtained in three independent experiments. (D) IP/Western blot with control, anti-ZYG11A, and anti-ZYG11B antibodies in U2OS cells. Gels were cut above the chicken IgY antibody heavy chain band. Similar results were obtained in two independent experiments. (E) IP of endogenous cyclin B1 coprecipitates endogenous ZYG11B in HeLa and HEK293T cells treated with MG132. Control (anti–VSV-G) and anti–cyclin B1 IPs were performed from the same initial cell lysate. Similar results were obtained in three to four independent experiments.