Figure 3.

Ligand tail is required for activation of the FKBP-FRB synNotch (ff-synNotch) system. (A) Schematic of the ff-synNotch system. Here, FKBP and FRB replace the N-terminal portion of Dll4 and the Notch1 EGF-like repeats 1–23, respectively.⁶ (B) Luciferase activity assay showing the activation of a Notch reporter cell line (U2OS-FRB-Notch1-Gal4 transfected with a UAS-Luciferase reporter) co-cultured with CHO-TetR cells expressing the indicated variants of FKBP-hDll4 ligand either with or without ligand ICD (FKBP-hDll4-ΔICD). (C) Images showing a co-culture of inducible CHO-TetR cells expressing the FKBP-hDll4 or FKBP-hDll4-ΔICD (red), with U2OS cells expressing the FRB-Notch-citrine (Citrine tag is inserted in the ECD, green). Images were taken 10 h after the induction of ligand expression with 100 ng/mL dox followed by 1 h induction of binding with 250 nM rapamycin. TEC is observed in full-length ligands (white arrows). Accumulation on the boundaries, but no TEC are observed in ligands lacking their ICD (orange arrows). Experiments with ligands lacking the ICD show reverse TEC (blue triangle). Data points show mean values from n = 13 measurements from four independent experiments. Error bars represent S.E.M. ***p < 0.001. Scale bars-10 μm.