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Fig. 5. EGFR ligands and EGFR act as an upstream regulator of tyrosine phosphorylation of p145^met, activation of Src and survival in serum-starved 5637 cells. (A) Normally grown 5637 cells (+ FCS) were treated with 125 ng/ml HGF (+ FCS/HGF), conditioned RPMI1640 media from 24-hour normally grown 5637 cells (Cond./+ FCS med), conditioned RPMI1640 media from 24-hour serum-starved 5637 cells (Cond./– FCS med), or fresh RPMI1640 medium containing either 10% FCS (Fresh/+ FCS med) or no FCS (Fresh/– FCS med) for 1 hour. After treatment, whole cell extracts (20 µg/lane) were prepared and analyzed by immunoblotting with either anti-phosphotyrosine antibody PY99 or anti-phosphoMAPK antibody. The positions of pp145^met, pp44, and pp42 are indicated. (B) Normally grown 5637 cells were treated with the indicated concentrations (0, 1 or 10 ng/ml) of EGF for 10 minutes in the absence or presence of 1 µg/ml anti-EGFR monoclonal antibody mAb528 or 1 µg/ml normal mouse IgG. After treatment, whole cell extracts were prepared and analyzed by immunoprecipitation (200 µg/lane) and/or immunoblotting (20 µg/lane for direct analysis) with the indicated antibodies. The positions of pp170, pp145, pp60^src and p170/EGFR are indicated. (C) Whole cell extracts were prepared from 5637 cells that had been treated with or without 10 ng/ml EGF for 10 minutes as in panel B, and subjected to immunoprecipitation (200 µg/lane) with either anti-EGFR or anti-Met antibodies followed by immunoblotting with either anti-phosphotyrosine antibody PY99, anti-EGFR or anti-Met antibodies. The positions of pp170, pp145, p170/EGFR and p145^met are indicated. (D) Carcinoma 5637 cells were treated with either normal medium (10% FCS) containing 125 ng/ml HGF for 1 hour or serum-free medium for 24 hours in the absence or the presence of 1.25 mM CuCl₂, 1.25 mM MgCl₂, 1 µg/ml anti-EGFR monoclonal antibody mAb528 or 1 µg/ml normal mouse IgG. After the treatments, whole cell extracts (20 µg/lane) were prepared and analyzed by immunoblotting with either anti-phosphotyrosine antibody PY99 or anti-Met antibody. The positions of pp145^met, p145^met and p180^met (asterisk) are indicated. (E) Carcinoma 5637 cells (1x10⁶ cells/dish) were cultured in normal conditions (10% FCS) for 48 hours, and then further cultured for an additional 48 hours in serum-free medium containing none (closed circles), 1 µg/ml anti-EGFR monoclonal antibody mAb528 (closed triangles) or 1 µg/ml normal mouse IgG (closed squares). Cell number was determined at 24, 48, 72 and 96 hour post initial treatment as in Fig. 1A. (F) Carcinoma 5637 cells were treated as in E. After treatment (96 hours post treatment), cell death (black bar) and caspase 3/7 protease activity (grey bar) of the whole cell extracts (20 µg/assay) were determined as in Fig. 1D. Data shown are mean ± s.d. of three independent experiments. *P<0.01 compared with levels in the control.

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