Fig 2: Flowchart of in vitro high-throughput screening and validation assay of candidate clones Flowchart of in vitro high-throughput screening.Validation of screening using CRBN mutant. Interaction between FLAG-GST-SALL4, FLAG-PLZF or IKZF1 and bls-CRB-WT or bls-CRBN-YW/AA in the presence of DMSO or 50 µM thalidomide was analysed by in vitro binding assay using the AlphaScreen technology. In vitro binding assay using pull-down and immunoblot analysis. bls-CRBN-WT or bls-CRBN-YW/AA was used as bait protein, and thalidomide-dependent interactions between bls-CRBN and FLAG-GST-SALL4, PLZF and IKZF were confirmed by immunoblot analysis after streptavidin pull-down. In vitro binding assay for ZBTB family proteins using the AlphaScreen technology. FLAG-GST-ZBTB proteins (ZBTB17, ZBTB20, ZBTB38, ZBTB39, ZBTB48 and PLZF) were evaluated for thalidomide-dependent interactions with bls-CRBN by same procedures indicated in Fig EV1B. In vitro binding assay for thalidomide, pomalidomide and lenalidomide. Interaction between bls-CRBN and FLAG-GST-SALL4 in the presence of (DMSO, 3.125, 6.25, 12.5, 25, 50, 100 or 200 µM) thalidomide, pomalidomide or lenalidomide was analysed using the AlphaScreen technology. Data information: All relative AS (AlphaScreen) signals were expressed as relative luminescent signal with luminescent signal of DMSO as one. The error bars denote ± standard deviation (n = 3). Source data are available online for this figure.

Fig 3: IMiD-induced protein degradation of PLZF in B cell lymphomas HT, TK, BJAB, SU-DHL-4 and MT-4 cells were treated with DMSO (DM), 10 µM lenalidomide (Le) or pomalidomide (Po) for 24 h. PLZF protein levels were analysed by immunoblot.Raji cells were treated with DMSO or pomalidomide for 24 h. PLZF protein levels were analysed by immunoblot. Source data are available online for this figure.

Fig 4: Downregulation of PLZF causes abnormal limb development in chicken embryo Sall4, Plzf or Crbn mRNA expression in E4 chicken embryos was analysed by whole-mount in situ hybridisation. Upper panel shows whole chicken embryo, and lower panel shows right forelimb bud. Sall4, Plzf or Crbn mRNA expression in E4 chicken limb bud was analysed by section in situ hybridisation.Immunoblot analysis of AGIA-GgPLZF in AGIA-GgPLZF expressing DF-1 cells transfected with shControl (shGFP) or shPLZF expression vector.Immunoblot analysis of PLZF from tissue of chicken forelimb or hindlimb bud. Endogenous PLZF protein expression was detected by immunoblot using E4.5 chicken embryos infected with RCAN virus packaging shControl or shPlzf (forelimb shControl [n = 4], forelimb shPLZF [n = 4], hindlimb shControl [n = 2] or hindlimb shPlzf [n = 3]).Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPLZF (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. Data in bar graphs were calculated as relative bone length with bone length of shControl as 100. Error bars denote ± standard deviation (forelimb and hindlimb shControl [n = 3], forelimb shPLZF [n = 5] or hindlimb shPLZF [n = 6]). P-values were calculated by one-way ANOVA with Tukey's post hoc test (**P < 0.01 and ****P < 0.0001).Teratogenic phenotypes of chicken embryos in Fig 6E. Source data are available online for this figure.

Fig 5: Interaction and protein degradation analyses between PLZF or SALL4 and CRBN with thalidomide among human, mouse and chicken A In vitro binding assay using human, mouse and chicken proteins. Thalidomide-dependent interaction between biotinylated HsCRBN, MmCRBN or GgCRBN, and FLAG-GST-SALL4 or FLAG-GST-PLZF (Hs, Mm or Gg) in the presence of DMSO or 200 µM thalidomide was analysed using the AlphaScreen technology.B, C In vitro binding assay using human or mouse proteins. Thalidomide-dependent interaction in the presence of DMSO or 50 µM thalidomide was analysed using the same procedure used in Fig EV5A.DImmunoblot analysis of AGIA-GgPLZF in FLAG-GgCRBN-WT or FLAG-GgCRBN-E379V expressing CRBN-/- HEK293T cells treated with DMSO, 50 µM or 200 µM thalidomide for 16 h. Data information: All relative AS (AlphaScreen) signals are expressed as a relative luminescent signal with a luminescent signal of DMSO as one. Error bars denote ± standard deviation (n = 3). P-values were calculated by one-way or two-way ANOVA with Tukey's post hoc test (NS = not significant, *P < 0.05, ***P < 0.001 and ****P < 0.0001). Source data are available online for this figure.