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. Author manuscript; available in PMC: 2021 Jul 6.
Published in final edited form as: Sci Transl Med. 2021 Jan 6;13(575):eabb6295. doi: 10.1126/scitranslmed.abb6295

Fig. 2: Engineering of a lenalidomide-inducible dimerization system and ON-switch split CAR.

Fig. 2:

Bioluminescence resonance energy transfer (BRET) was used to measure the association between the indicated NanoLuc luciferase and HaloTag fusion proteins in 293T cells. (A) BRET analysis of IKZF3 interaction with CRBN deletion variants. IKZF3 = amino acids (AA) 130–189 of IKZF3. NTD, N-terminal domain; LLD-N, Lon-like domain (N-terminal portion); LLD-C, Lon-like domain (C-terminal portion); TBD, thalidomide-binding domain. (B) BRET analysis of IKZF3-CRBNΔ3 incorporated into cell surface-localized fusion proteins. 1928 = FMC63 scFv – CD28 hinge, transmembrane, and costimulatory domains. CD8-CD28 = CD8 hinge and transmembrane domain and CD28 co-stimulatory domain. PD1 = PD1 transmembrane and cytoplasmic domain. LYN-CD28 = LYN myristoylation and palmitoylation motif – CD28 costimulatory domain. MG132 was included in experiments A and B. (C) BRET analysis of CD8-CD28-CRBNΔ3 and 1928-IKZF3 with or without intracellular Lys → Arg mutations (iK0). mBU, miliBRET units. Individual values and nonlinear regression are shown. Data represent one independent experiment performed in technical duplicate. Two-way ANOVA, **** P < 0.0001.