preTCR complex is rapidly internalized from cell surface. (A) Schematic demonstrating the brefeldin A (BfA) assay, where anterograde flow of newly formed receptors is blocked, so that the internalization rate of surface-expressed receptors can be measured in isolation. (B) HEK cells expressing either the αβTCR or preTCR were incubated for a defined period with either BfA or DMSO vehicle control before receptors still present at plasma membrane were detected with an anti-TCR β antibody. The decrease in intensity from the initial time point follows the sustained internalization of the receptors. Data points show mean ± SEM (n = 3); asterisks indicate P < 0.01 when comparing αβTCR and preTCR datasets in presence of BfA. (C) Data from one representative BfA assay, where the internalization rate is shown at different binned expression levels based on the range of transfection in HEK cells. (D) Repeat of the BfA assay as in B but also performed with HEK cells coexpressing AP180C, which disrupts clathrin-mediated endocytosis. Datapoints show mean ± SEM (n = 3); asterisks indicate P < 0.05 when comparing effect of AP180C on preTCR datasets in presence of BfA. A two-tailed, two-sided t test was used for all statistical analyses.