Extended Data Figure 2: CC-885, but not lenalidomide, promotes the interaction between GSPT1 and CRBN in vitro.

a, b, Immunoblot analysis of anti-HA immunoprecipitates (a) and whole-cell lysates (WCL) (b). HA-tagged GSPT1 or IKZF1 produced in 293FT CRBN^−/− HEK cells were used to capture CRBN from 293FT HEK cells expressing GSPT1-specific shRNA, shGPST1-1. DMSO, 10 μM lenalidomide or 10 μM CC-885 were included in the binding assay. The IKZF1 Q146H mutation results in a reduction of cereblon binding mediated by either lenalidomide or CC-885. Some residual binding is observed with CC-885, consistent with Fig. 1d, which shows that CC-885 is more potent than lenalidomide against IKZF1. c, Coomassie stain of CRBN–DDB1 pull-down with GSPT1 using purified components. Purified GST or GST–GSPT1 domains 2 and 3 (amino acids 437–633) bound to magnetic glutathione beads was incubated with purified CRBN–DDB1 in the presences of CC-885, lenalidomide (len), pomolidamide (pom), or DMSO (vehicle) for 1 h at room temperature before three rapid washes. CC-885, but not lenalidomide, pomalidomide or DMSO, mediated the binding of GST–GSPT1 to CRBN–DDB1. This experiment was performed three times. For gel source data, see Supplementary Information Fig. 1.