Figure 5.

EGFR and Met association independent of EGFR kinase activity. (A) SUM149 or SUM229 cells were treated with 0.5 μM gefitinib or DMSO control for 30 min. Cells were lysed and immunoprecipitated with anti-EGFR, anti-Met, and anti-IgG. Whole cell lysates were used as a control. Immunoprecipitates were separated by SDS-PAGE, transferred, and immunoblotted using anti-EGFR, anti-Met, or anti-c-Src antibodies. (B) SUM229 cells were incubated with EGFR shRNA lentiviral particles for 72 hrs. Lysates were prepared and separated by SDS-PAGE. Membranes were immunoblotted with anti-pMet, anti-Met, anti-EGFR, and β-actin. (C) SUM229 cells were treated with increasing concentrations of dasatinib for 2 hrs. Lysates were prepared and immunoblotted with anti-pMet, anti-Met, and anti-pSrc antibodies. (D) SUM229 cells were incubated with EGFR shRNA lentiviral particles for 24 hrs and then treated with puromycin to select for virus expressing cells. After the puromycin was added for 24 hrs, the cells were grown for 7 days at which point cell numbers were determined via Coulter counting.