Anti her3

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®).

The reagents used were as follows: epoxomicin (Peptide); ethanol, cycloheximide and chloroquine diphosphate (Wako); dimethyl sulfoxide and Fulvestrant (Sigma-Aldrich). The antibodies used were as follows: anti-HER3, anti-NEDD4-1 and corresponding secondary antibodies (Cell Signaling Technology); anti-Itch and anti-Nrdp1 (Santa Cruz Biothechonology); anti-ER (Thermo Scientific): anti- β actin (Sigma-Aldrich).

Anti-HMGA1 (ab129153 and ab4078, Abcam, Cambridge, UK), anti-E2F1, anti-Lamin A and anti-PCNA (KH-95, H-102 and PC-10 Santa Cruz Biotechnology, CA, USA), anti-PARP (#9548), anti-Zeb1 (#3396), anti-Vimentin (#5741), anti-pHER3 (#4791), anti-HER3 (# 12708), anti-HRGβ1 (#2573), anti-pIKKα/β (#2697), anti-IKKα (#11930), anti-pP65 (#3033), anti-P65 (#8242), anti-IKB (#4814) were from Cell Signaling (Danvers, USA), and anti-Hsp-70 (ab-83392) was from Immunological Sciences (Rome, Italy). HRP-conjugated secondary antibodies were from Bio-Rad (CA, USA).

Western blotting was performed as previously described [31 (link)]. The antibodies used were as follows: monoclonal anti-JWA (1 : 500, contract produced by AbMax, Beijing, China); monoclonal anti-α-tubulin, anti-β-actin (loading control) (1 : 2000, Beyotime, Haimen, Jiangsu, China); monoclonal anti-P-ERK, anti-ERK, anti-P-AKT(473), anti-AKT, anti-Caspase 3, anti-EGFR, anti-HER3 (1 : 1000, Cell Signaling Technology, Danvers, MA, USA); monoclonal anti-Ub (1 : 500) and polyclonal anti-HER2 (1 : 1000) (Santa Cruz, Dallas, TX, USA); monoclonal anti-c-Cbl (1 : 2000), anti-Lamp2 and anti-HER2 (1 : 250, Abcam, USA).

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.