preTCR internalization requires all complex components and is not constitutively phosphorylated in HEK cells. (A) The internalization assay was applied to HEK cells expressing HA-tagged pTa protein chain in the presence of defined components of the preTCR complex. An AF647-conjugated anti-HA antibody was used to quantify potential pTa internalization. Schematics above each plot show the chains expressed in HEK cells. Only in the presence of all preTCR components can the anti-HA antibody become internalized. One representative experiment from two replicates is shown. (B) HEK cells expressing the preTCR complex or additionally with LCK or ZAP70 kinases as denoted, were lysed and subjected to Western analysis. Blotting with the indicated antibodies shows that only in the presence of exogenously expressed kinases is CD3 ζ-chain phosphorylation detectable. One representative experiment from two replicates is shown. Source data are available for this figure: SourceData FS2.