The huEGFRt expressed on selected T cells is inert. (A) EGF does not bind to the surface of huEGFRt-expressing T cells. A431, EGFRt-selected T cells, and negative control T cells were analyzed by flow cytometry after incubation with PE-conjugated anti-EGFR, or either biotinylated cetuximab or biotinylated EGF followed by PE-conjugated streptavidin (black histogram) versus PE-conjugated isotype control antibody or streptavidin alone (gray histogram). Percentage positive staining is indicated in each histogram. (B) huEGFRt expressed on T cells is not phosphorylated on coincubation with EGF. Negative control T cells, huEGFRt-selected T cells, or A431 cells were incubated for 5 minutes with or without 100 ng/mL EGF and then lysed in the presence of phosphatase inhibitor. Lysates run on Western blots were then probed using antibodies specific for β-actin, the cytoplasmic domain of EGFR, or the phosphorylated tyrosine at position 1068 of EGFR.