Total anti egfr

2x10⁶ cells were grown for 72h then serum-starved for 48h in a phenol red-free medium in presence or absence of different concentrations of 5x10⁻⁶ M SR 48692 and 25x10⁻⁹ M MMP9 inhibitor (Calbiochem®). For EGF treatments, cells were then treated 15 min with EGF (10 ng/mL) lysed (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 0,5% NP40, 0,5% glycerol, 1 mM PMSF, protease and phosphatase inhibitor cocktail) at 4°C for 30 min. Samples in Laemmli buffer were loaded on 10% SDS-PAGE and transferred to PVDF membranes. Saturation was performed 30 min at room temperature in 5% non-fat dry milk in TBS 0.1% Tween 20. Primary antibodies were incubated overnight at 4°C according to the manufacturer's instructions. Total anti-EGFR (1:500), anti-phospho-EGFR (1:500), anti-phospho-HER2 (1:500), anti-HER3 (1:2000), anti-phospho-HER3 (1:1000), anti-ERK 1/2 (1:2000) were from Cell Signaling Technology. Total anti-HER2 (1:2000) were purchase from Neomarkers and anti-βactin (1:50000) from Sigma. Secondary anti-rabbit (Santa Cruz Biotechnology®) or anti-mouse (Sigma®) antibodies conjugated to HRP were used at 1:2000 dilutions for 1h at room temperature and visualised by enhanced chemiluminescence (GE Healthcare®).

2×10⁶ cells were grown for 72h then serum-starved for 48h in a phenol red-free medium in presence or absence of different concentrations of 5×10⁻⁶ M SR 48692 and 25×10⁻⁹M MMP inhibitor (Calbiochem), and lysed (20 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgCl2, 0,5 % NP40, 0.5 % glycerol, 1 mM PMSF, protease and phosphatase inhibitor cocktail) at 4°C for 30 min. Primary antibodies were incubated overnight at 4°C according to the manufacturer's instructions. Total anti-EGFR (1:500), anti-phospho-EGFR (1:500), anti-phospho-HER2 (1:500), anti-HER3 (1:2000), anti-phospho-HER3 (1:1000) were from Cell Signaling Technology. Total anti-HER2 (1:2000) was purchased from Neomarkers and anti-βactin (1:50000) from Sigma. Secondary anti-rabbit (Santa Cruz Biotechnology) or anti-mouse (Sigma) antibodies, conjugated to HRP, were used at 1:2000 dilutions for 1h at room temperature and visualised by enhanced chemiluminescence (GE Healthcare).

Samples in Laemmli buffer were loaded on 10% SDS-PAGE and transferred to PVDF membranes. Membranes were exposed to primary antibody overnight. Total anti-EGFR (1:500), anti-phospho-EGFR (1:500), anti-phospho-HER2 (1:500), anti-HER3 (1:2000), anti-phospho-HER3 (1:1000), anti-ERK 1/2 (1:2000) were from Cell Signaling Technology®. Total anti-HER2 (1:2000) were purchased from Neomarkers and anti-βactin (1:50000) from Sigma®. Secondary anti-rabbit (Santa Cruz Biotechnology) or anti-mouse (Sigma®) antibodies conjugated to HRP were used at 1:2000 dilutions for 1h at room temperature and visualized by enhanced chemiluminescence (GE Healthcare®). See details in supplementary information.