Figure 6.
Exposed extracellular preTCR domain drives constitutive internalization. (A) Schematic showing the various receptor variants used in the figure. The exposed hydrophobic region (Φ) is marked in red. The intracellular region of CD3ζ chain has been removed for clarity but was present in all experiments. (B) Surface staining for TCRβ of receptors indicated in legend as a function of expression, with staining performed at 4°C to inhibit internalization. The intracellular sequence of preTCR (“Tail”) has no effect on constitutive surface expression. Data is shown as bounding area around datapoints with mean ± SEM (n = 3). (C) Equivalent datasets as in B but receptor-transfected HEK cells were incubated with anti-TCR antibody at 37°C for 30 min to identify any effect of the preTCR tail on the rate of internalization. Data is shown as a bounding area around data points with mean ± SEM (n = 3). (D) Surface staining for TCRβ of receptors indicated as a function of expression, with staining performed at 4°C to inhibit internalization. Data are shown as bounding area around datapoints with mean ± SEM (n = 3). (E) All datasets from D overlaid for direct comparison. (F) Brefeldin A (BfA) assay to measure the internalization rate of the indicated receptor variant, with vehicle control shown with open circles. Datapoints show mean ± SEM (n = 3). (G) Datasets from F overlaid with previous BfA assay datasets for αβTCR and preTCR complexes, which were collected at the same time to allow direct comparison. Datapoints show mean ± SEM (n = 3).

Exposed extracellular preTCR domain drives constitutive internalization. (A) Schematic showing the various receptor variants used in the figure. The exposed hydrophobic region (Φ) is marked in red. The intracellular region of CD3ζ chain has been removed for clarity but was present in all experiments. (B) Surface staining for TCRβ of receptors indicated in legend as a function of expression, with staining performed at 4°C to inhibit internalization. The intracellular sequence of preTCR (“Tail”) has no effect on constitutive surface expression. Data is shown as bounding area around datapoints with mean ± SEM (n = 3). (C) Equivalent datasets as in B but receptor-transfected HEK cells were incubated with anti-TCR antibody at 37°C for 30 min to identify any effect of the preTCR tail on the rate of internalization. Data is shown as a bounding area around data points with mean ± SEM (n = 3). (D) Surface staining for TCRβ of receptors indicated as a function of expression, with staining performed at 4°C to inhibit internalization. Data are shown as bounding area around datapoints with mean ± SEM (n = 3). (E) All datasets from D overlaid for direct comparison. (F) Brefeldin A (BfA) assay to measure the internalization rate of the indicated receptor variant, with vehicle control shown with open circles. Datapoints show mean ± SEM (n = 3). (G) Datasets from F overlaid with previous BfA assay datasets for αβTCR and preTCR complexes, which were collected at the same time to allow direct comparison. Datapoints show mean ± SEM (n = 3).

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