Substrate degradation is dependent on a limiting pool of CRBN. (A) MM1S cells that constitutively express CAS9 were transduced with lentivirus-expressing single guide RNAs and a fluorophore (tagRFP or RFP657). Data are mean ± SEM (n = 3 replicates). (B) MM1S cells were transduced with retrovirus-expressing GFP and CRBN. Relative growth was assessed by means of flow cytometry after treatment with decreasing doses of lenalidomide (Len) or vehicle. Data are representative of 2 independent experiments. (C-D) Levels of IKZF3 and CK1α protein as measured by the iMRM assay following treatment with 1 µM lenalidomide in MM1S cells overexpressing CRBN (CRBN OE; n = 3 replicates). Protein levels were normalized to a housekeeping factor and the level at the start of the experiment was normalized to 1. (E-F) Levels of IKZF3 and CRBN protein as measured by the iMRM assay in each of the 10 MM cell lines after treatment with 1 µM lenalidomide or vehicle for 24 hours. Protein levels were normalized to the housekeeping factor (n ≥ 3 replicates). (E) The ratio of IKZF3 in the lenalidomide-treated as compared with vehicle-treated samples is plotted against the absolute CRBN level in vehicle-treated samples. Points marked in red represent lenalidomide-sensitive cell lines. (F) Protein level of CRBN in each of the cell lines after treatment with 24 hours of 1 µM lenalidomide or vehicle. P values from a 2-sided Student t test. Data are mean ± SEM (n ≥ 3 replicates). NTG, nontargeting guide.