Figure 1

Met expression mediates EGFR tyrosine phosphorylation and proliferation in the absence of EGFR kinase activity. (A) SUM229 cells were infected with lentiviral particles containing Met shRNA or a non-silencing shRNA for 72 hrs. Cells were treated with gefitinib for the last hour of infection at 0.5 μM, cells were lysed, and lysates were immunoprecipitated with anti-EGFR. Immunoprecipitates were immunoblotted with anti-EGFR, anti-Ptyr, and the indicated phospho-specific sites on the EGFR. Corresponding whole cell lysates were immunoblotted with anti-Met and β-actin as a loaded control. Through quantification, EGFR phosphorylation was decreased by 53% in the gefitinib treated cells and 88% in the gefitinib with Met knocked down cells. The two bands in the Met immunoblot represent processed and pro-forms of Met. (B) SUM229 cells were infected with lentiviral particles containing Met shRNA in the presence of gefitinib or a non-silencing control for seven days in the presence of puromycin to select for infected cells. Cells were counted using a Coulter Counter and the day 8 values were graphed with the error bars representing SEM.