IMiDs interfere with the direct binding of THBS-1 and cereblon. (A and B) Protein expression of THBS-1 (A) and relative mRNA expression levels of THBS-1 (B) in cereblon-depleted MEG01 cells. In panel B, data are from 3 independent experiments and expressed as means ± SEM. P values were determined by 2-tailed Student t test. (C) Immunoblot analysis for THBS-1 in cereblon-depleted MEG01 cells treated with DMSO, 10 μM lenalidomide, or 10 μM pomalidomide for 24 hours. (D) Lysate mixture of MEG01 cells and 293T cells transfected with FLAG-tagged cereblon were incubated with or without 10 μM lenalidomide and subjected to immunoprecipitation with antibody against FLAG. (E) Purified recombinant His-tagged THBS-1–loaded beads were incubated with glutathione S-transferases–tagged cereblon in the presence of pomalidomide at indicated concentration. Mixture was immunoprecipitated with antibody against His. Results are representative of 3 independent experiments (panels A, C, D, and E). (F) Homologous sequences in THBS-1 and cereblon binding site of IKZF1 are aligned. The identical residues are highlighted in red. Residues of IKZF1 composing backbone of zinc-finger structure are shown in bold characters. Arrows represent indicated interacting residues between IKZF1 and cereblon in previous report. Sequence similarity with each protein was analyzed using the protein-protein BLAST program on The National Center for Biotechnology Information. CRBN, cereblon; Cont, control; GST, glutathione S-transferases; IB, immunoblotting; IP, immunoprecipitation; Len, lenalidomide; Pom, pomalidomide; Thal, thalidomide.