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. 2005 Dec;7(12):1065–1072. doi: 10.1593/neo.05553

Figure 3.

Figure 3

The CD3-EGFR chimeric receptor is sensitive to the EGFR tyrosine kinase inhibitor PKI166. (A) The kinase activity of HEK cells expressing wtEGFR (lanes 1–3) and CD3-EGFR (lanes 4–6) is inhibited by PKI166. The expression plasmid for wtEGFR or CD3-EGFR (1 µg) was transfected into HEK cells. wtEGFR-transfected cells were treated with EGF (40 ng/ml) alone or in combination with PKI166 (1 µM) for 60 minutes. CD3-EGFR-transfected cells were treated with 1 µM (+) or 5 µM (++) PKI166 for 60 minutes. Total cellular protein extracts were prepared to analyze expression and phosphorylation at specified tyrosine residues of wtEGFR and CD3-EGFR. Tyrosine-phosphorylated and total Stat3 protein levels are also shown. (B) Suppression of the constitutive activity of CD3-EGFR by PKI166 in human melanoma SB2 cells. Parental SB2 cells (lanes 1 and 4) and SB2 cells stably transduced with either CD3ζ (lanes 2 and 5) or CD3-EGFR (lanes 3 and 6) were used. Untreated cells (lanes 1–3) or cells treated with 1 µM PKI166 (lanes 4–6) for 60 minutes were prepared and analyzed for CD3-EGFR phosphorylated at Y1068 and total CD3-EGFR. Actin was used as a loading control.