Effects of exon 8 deletion on CRBN-mediated IMiD activity and interaction with DDB1. (A) Using site-directed mutagenesis by PCR, we generated the CRBN with exon 8 and 10 deletion alone. There were expressed in OCIMY5 and detected by western blot. (B-C) MTT assay demonstrated that exon 8 deletion alone, like exon 10 deletion, cannot mediate pomalidomide- and CFT13778-induced antimyeloma activity. (D-E) co-IP experiment was performed on untreated (−) or lenalidomide treated (+) OCIMY5 cells overexpressing WT and exon 8–deleted CRBN and it demonstrated that exon 8 deletion impaired the binding of CRBN to DDB1 and Cul4A. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Vec = vector, WT = wild type, Co-IP = co-immunoprecipitation.