Fig. 4.

Inhibition of EGFR reduces phosphorylation, association with SETA and Cbl, and polyubiquitination. CHO cells were treated with AG1478, and, if transfected with EGFR, were also stimulated with EGF for 5 min. EGFR or ΔEGFR-specific IPs were prepared as appropriate and showed that, with increased dosage of AG1478, the level of phosphotyrosine on EGFR was reduced, as was its binding to SETA and Cbls. In addition, polyubiquitination of the EGFR, visible as an HA-positive smear, was suppressed by AG1478. (Lower) Quantification of the phosphotyrosine signal relative to the EGFR signal is shown, with the values for untreated EGFR being set arbitrarily to 1 in each case. The phosphorylation level of ΔEGFR is similar to that of EGFR from cells treated with 4 nM AG1478. This concentration is the start of the threshold at which levels of SETA and Cbl proteins in EGFR complexes declined.