Equivalent ER export is observed for the αβTCR and preTCR complexes. (A) Schematic showing RUSH assay applied to preTCR ER export. SBP-GFP fused to the cleaved N-terminus of TCRβ causes ER retention through binding to streptavidin (SAKDEL). Biotin addition causes the release of this lock and synchronous export of complexes from ER, where the receptors can bind extracellular anti-GFP nanobody (Nb) and internalize it into endosomes. (B) Selected images showing the binding and internalization of anti-GFP Nb when SBP–αβTCR complexes are released from ER on biotin addition. Colored boxes denote protein representation in the overlay image. Scale bar, 20 μm. (C) Equivalent images as in B but for the preTCR complex when released from the ER. (D) Quantification of vesicles containing internalized anti-GFP Nb when SBP–αβTCR is released on biotin addition (red). Equivalent data for αβTCR without SBP (black) is shown, highlighting the rate of Nb internalization without retention. Control data for SBP–αβTCR without biotin is also presented (green). Bounding area around datapoints shows mean ± SEM of multiple fields of view from six biological replicates; asterisks indicate P < 0.01 when comparing αβTCR datasets with biotin addition or not. (E) Equivalent quantification as in D for the SBP-preTCR (blue) RUSH assay. (F) Biotin addition plots from D and E normalized to detected particles at 75 min. (G) The correlation coefficient between the ER marker and receptor image channels with time is plotted for both αβTCR (red) and preTCR (blue) biotin addition datasets. The bounding area around data points shows mean ± SEM of multiple fields of view from six biological replicates. A two-tailed, two-sided t test was used for all statistical analyses.
