preTCR complex is constitutively trafficked to lysosomes. (A) Schematic describing the localization of internalized receptors to distinct parts of the endosomal pathway. GFP-fused receptors localized at the cell surface can bind anti-GFP nanobody (Nb) and internalize the fluorescently labeled Nb. Cotransfection with labeled RAB5A/RAB7A and LAMP1 constructs allows the identification of internalized receptor compartment with time. (B) Representative images from the colocalization assay for the αβTCR and preTCR complexes internalizing Nb with time. Colored boxes denote protein representation in the overlaid images. Scale bar, 10 μm. For all inset images, the bounding area is 8 μm in width. (C) Quantification of colocalization of internalized Nb-bound receptors with early (RAB5A) or late (RAB7A) endosomes and lysosomes (LAMP1). Data points show mean colocalization from three independent experiments with individual values and kernel density for the complete dataset also presented. The line shows the overall mean of combined experiments. (D) Datasets from C were combined to allow for direct comparison between αβTCR and preTCR complexes. Upper panels show mean ± SEM (n = 3 or 6) and lower panel shows t test statistic (P) comparing αβTCR and preTCR datasets. Dashed lines indicate P = 0.05. (E) Representative image showing colocalization of internalized Nb-bound αβTCR with RAB11A, a marker of recycling endosomes. Arrows mark exemplar colocalized spots and colored boxes denote protein representation in the overlaid image. Scale bar, 5 μm. (F) Quantification of colocalization of internalized Nb-bound receptors with recycling (RAB11A) endosomes, measured 5–20 min are Nb addition. Data points show mean colocalization from three independent experiments with individual values and kernel density for the complete dataset also presented. Asterisks indicate P < 0.01 when comparing αβTCR and preTCR datasets. A two-tailed, two-sided t test was used for all statistical analyses.