Fig. 4: Fluorophore phase separation-based assay for imaging ternary complex formation in living cells.

a Schematic diagram showing the design of the cellular assay. To detect PROTAC-induced PPI between BTK and CRBN, we engineered the kinase domain of BTK (amino acid residues 382–659, referred to as BTK^KD) into SPPIER to produce a BTK^KD-EGFP-HOTag6 construct, which forms tetramers when expressing in cells. The previously reported CRBN-EGFP-HOTag3 fusion construct, which forms hexamers in cells, was used as the E3 ligase SPPIER. If PROTACs can induce {BTK-PROTAC-CRBN} ternary complex formation in cells, they will crosslink the BTK^KD-EGFP-HOTag6 tetramers and the CRBN-EGFP-HOTag3 hexamers to produce EGFP phase separation, which can be conveniently visualized with a fluorescence microscope. b Fluorescence images showing detection of BTK PROTACs-induced interaction between the E3 ligase cereblon and the target protein BTK. A fluorescence histogram of the line across the cells is shown below. HEK293 cells transiently expressed BTK^KD-EGFP-HOTag6 and CRBN-EGFP-HOTag3. RC-1, IRC-1, and RNC-1 (10 μM) were added to the cells. Scale bar: 10 μm. Source data are provided as a Source Data file.