Anti human met

Antibodies used were from Cell Signaling: anti-Met 25H2 (1:2000 for WB), anti-phospho-Tyr_1234/1235-Met (1:2000 for WB; 1:50 for IHC), anti-phospho-Tyr₁₀₀₃-Met (1:2000 for WB), anti-phospho-Tyr₁₃₄₉-Met (1:1000 for WB), anti-phospho-Tyr₆₂₇-Gab1 (1:2000 for WB), anti-phospho-Ser₄₇₃-Akt (1:2000 for WB; 1:20 for IHC), anti-phospho-Ser₇₂₇-Stat3 (1:2000 for WB), anti-phospho-Thr₂₀₂-Tyr₂₀₄-ERKs (#9106; 1:10000 for WB), anti-phospho-Thr₂₀₂-Tyr₂₀₄-ERKs (#4376; 1:150 for IHC), anti-ERKs (1:10000 for WB); from Santa-Cruz Biotechnology: anti-mouse Met (1:200 for WB), anti-human Met (1:1000 for WB), anti-HGF (1:500 for WB); from Abcam: anti-β-galactosidase (1:2000 for WB); from R&D: anti-mouseHGF (1:50 for IHC); from Sigma-Aldrich: anti-actin (1:12000 for WB); from Assay Designs: anti-human Met (1:500 for IHC); from hybridoma bank: Pax3 (1:10 for IHC), anti-myosin heavy chain II (MF20; 1:50 for IHC); from Jackson: anti-rabbit IgG-peroxidase or anti-mouse IgG-peroxidase (1:4000 for WB), anti-mouse fluorescent-coupled secondary antibodies (1:400 for IHC), anti-mouse or rabbit biotin-coupled secondary antibodies (1:500 for IHC).

For immunohistochemical analysis, formalin‐fixed, paraffin‐embedded tissue sections were used for validation of immunostaining with anti‐human MET (1:300, Abcam, Cambridge, UK) and anti‐human CD24 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA). Immunohistochemistry (IHC) was performed according to standard IHC techniques. Membranous staining of MET and cytoplasmic staining of CD24 were considered positive and evaluated by light microscopy. Three different scales defined immunohistochemical staining results: (1) ‘Volume’, the proportion of stained cells; (2) ‘Volume × intensity’, staining intensity was classified as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) and the proportion of stained cells was multiplied by the staining intensity; and 3) ‘The modified score’ was determined as follows: 0, when less than 5% of tumour cells were weakly stained; 1, when 5%–50% of tumour cells were stained with any intensity or less than 5% of tumour cells were moderately or strongly stained; 2, when more than 50% and equal to or less than 90% more than 50% of tumour cells were stained with any intensity; and 3, when more than 90% of tumour cells were stained with any intensity. The modified score was regrouped for statistical analysis into negative (0, 1) and positive (2, 3).