MHC expression on HEK cells does not affect preTCR internalization. (A) Confirmation of complete MHC-I knockout in HEK cells. Wildtype (WT) HEK cells (blue) stained with AF647-labeled anti-MHC antibody show high expression of MHC that is completely absent on MHC-knockout cells (MHC KO; red) and is indistinguishable from isotype control staining (filled grey). (B) preTCR expression in either WT HEK cells or MHC KO cells shows no substantial difference in surface staining with APC-conjugated anti-V _β5.1 TCR antibody. (C) Repeating the preTCR internalization assay with WT and MHC KO HEK cells shows no substantial difference in the absence of MHC at any timepoint measured. One representative experiment from two replicates is shown.