preTCR complex shows no evidence for oligomerization at the cell surface. (A) Schematic showing the fusion of the NanoBiT components to the intracellular sequences of the preTCR complex, together with αβTCR positive control constructs. (B) Luminescence from NanoBiT assay of indicated receptor constructs and controls, relative to the value for the αβTCR positive control. Datapoints for each of the three biological replicates are shown. (C) Luminescence from NanoBiT assay with component BiTs now localized to the extracellular side of the indicated receptors. Values are shown relative to the αβTCR positive control, with data points for each of three biological replicates shown. (D) Schematic showing the trimolecular fluorescence complementation assay, assuming the preTCR complex is multivalent. Reformation of the split Venus fluorophore through receptor dimerization affords labeled nanobody binding at cell surface, which can be internalized with time and detected by flow cytometry. (E) Flow cytometry plots showing the relationship between the reformed Venus fluorophore and nanobody binding. Positive control (α ^VNβ^VCTCR) shows potent mVenus expression and concomitant nanobody binding, which is entirely absent from the preTCR sample. (F) Histogram showing nanobody binding at constitutive expression levels. (G) Representative datasets of nanobody binding to transfected HEK cells at 37°C with time, to allow potential internalization of undetectable receptor oligomers to be amplified. (H) Three biological replicates of datasets in G are quantified, with nanobody (Nb) staining shown with time and relative to the value measured for positive control.