Phosphorylation, Immunoprecipitation, and Western Blotting

These steps were conducted as previously described (23, 25). The phosphorylation assay was performed by stimulating BxPC3 cells (2 × 10⁶ cells/mL/sample) with 2 nM MSP (RON activation) and 2 nM HGF (MET activation), followed by TKIs (5 μM BMS777607, 5 μM INCB28060, 5 μM PHA665752, 0.5 μM Tivantinib) at 37°C for 60 min (25). Cellular proteins from cell lysates (30 μg per sample) and tissue lysates (50 μg per sample) were separated in 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) under reduced conditions. RON, MET, or other signaling proteins were detected by western blotting using R5029, ab51067, or the corresponding antibodies, visualized using enhanced chemiluminescence reagents and were analyzed using the VersaDoc MP 5000 Imaging system (Bio-Rad). The membranes also were reprobed with antibodies to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) to ensure equal sample loading.

For immunoprecipitation, cellular proteins (250 μg per sample) were mixed with anti-phospho-tyrosine PY-100 (1:100) coupled to protein G Sepharose beads. Proteins were separated in 8% SDS-PAGE under reduced conditions. Phospho-RON or phospho-MET was detected by western blotting using R5029 or ab51067.