Anti phospho c met

The expression and phosphorylation of c-Met were evaluated by western blotting. Cells were washed with ice-cold phosphate buffered saline (PBS) and lysed with lysis buffer (Cell signaling Technology, Danvers, MA, USA) and protease and phosphatase inhibitors as follows: aprotinin (10 mg/mL), leupeptin (10 mg/mL) (ICN Biomedicals, Asse-Relegem, Belgium), phenylmethylsulfonyl fluoride (1.72 mM), NaF (100 mM), NaVO3 (500 mM), and Na4P2O7 (500 mg/mL) (Sigma-Aldrich). The protein concentration was determined by BCA assay (Pierce, USA). Equal amounts of protein were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred onto polyvinylidene fluoride (PVDF) membrane. Blots immunostaining was performed using the primary antibodies, followed by secondary antibodies conjugated to horseradish peroxidase, and detected by enhanced chemiluminescence reagent (Pierce, USA). The primary antibodies used were anti-phospho-c-Met (pY1230+Y1234+Y1235), anti-phospho-c-Met (pY1349), and c-Met (Epitomics; CA, USA). The secondary antibodies were purchased from Amersham Biosciences (Piscataway, NJ, USA).