Validation of IKZF1 and IKZF3 as CRBN-binding proteins. (A) Immunoblotting of the Ni⁺ beads pull-down proteins from OCI-MY5 vector (control) and OCI-MY5 CRBN-His with (+) without (–) lenalidomide treatment of 48 hours (2 independent experiments). CRBN, DDB1, CUL4A, IKZF1, IKZF3, and KPNA2 showed similar changes detected by MS analysis. (B-C) OCI-MY5 CRBN-His and MM1.S cells were treated with lenalidomide (10 μM) in a time course, and the changes of some top proteins identified from MS analysis or related with lenalidomide-induced cytotoxicity were measured by western blot. IKZF1 and IKZF3 downregulation was identified as the earliest change after lenalidomide treatment. (D) OCI-MY5 CRBN-His cells were treated with dimethyl sulfoxide or lenalidomide for 3 hours, and Co-IP was performed with anti-CRBN antibody to immunoprecipitate CRBN and CRBN-associated proteins, using a normal mouse IgG as a negative control. IKZF1 and IKZF3 were greatly increased in CRBN antibody–immunoprecipitated samples after lenalidomide treatment (left panels), but they are decreased in cell lysate after lenalidomide treatment (right panels). CRBN increased in both immunoprecipitated and nonimmunoprecipitated lysate.