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. 2022 Sep 15;11(10):3343–3353. doi: 10.1021/acssynbio.2c00247

Figure 4.

Figure 4

Type of TM domain does not affect dependence on ligand ICD. (A) Schematic of the constructs used for testing the effect of the TM region on Notch activity. (B) Luciferase activity assay showing the activation of a Notch reporter cell line (U2OS-Notch1-Gal4 or U2OS-FRB-Notch1-Gal4 transfected with a UAS-Luciferase reporter) co-cultured with CHO-TetR cells expressing the indicated variants either with or without ligand ICD. (C) Images showing a co-culture of inducible CHO-TetR cells expressing the ligand variants (red) with U2OS cells expressing the Notch1-Citrine or FRB-Notch1-Citrine (green). Images were taken 10 h induction of ligand expression with 100 ng/mL dox followed by 1 h induction of binding with 250 nM rapamycin (only for the ff-synNotch system). TEC was observed with full-length ligands (white arrows). Accumulation at the boundaries of cell contact without TEC was observed with ligands lacking their ICD (orange arrows). Experiments with ligands lacking the ICD show reverse TEC (blue triangle). Data points show mean values from n = 14 measurements from four independent experiments. Error bars represent S.E.M. ***p < 0.001. Scale bars-10 μm.