Extended Data Figure 3: Sequence determinants of CK1α degradation.

a, 293T cells were transfected with plasmids expressing Flag–CK1α isoform 1 or isoform 2 together with a human CRBN-expressing plasmid. Cells were treated with DMSO or 10 µM lenalidomide for 16 h. Cells expressing Flag–CK1α isoform 1, which contains a nuclear localization domain, were incubated in the absence or presence of the nuclear export inhibitor leptomycin B. b, 293T cells expressing Flag–CK1α isoform 2 wild-type or two different point mutations identified in patient samples were treated with DMSO or 10 µM lenalidomide for 16 h. c, Immunofluorescence for CK1α after treatment with DMSO or 10 μM lenalidomide. Enlarged area is indicated by a box in Merge. FITC channel represents staining for CK1α. No changes in CK1α localization are seen upon lenalidomide treatment. Experiment was performed twice in biological duplicate. In each condition, at least 25 cells were assessed. d, Chimaeric proteins of casein kinase 1A1 (CK1α) and casein kinase 1E (CK1ε), which shares significant homology with CK1α but is not responsive to lenalidomide, that were used in e to determine the lenalidomide-responsive region in CK1α. e, Flag-tagged (chimaeric) proteins from d were transfected in 293T cells together with a CRBN-expressing plasmid. Cells were treated with 1 µM lenalidomide for 24 h and protein was detected with a Flag-specific antibody. Data are representative of two (a, c), three (b) or four (e) independent experiments. Uncropped blots are shown in Supplementary Fig. 1.