P raf

The HRVECs were lysed with ice-cold RIPA buffer and the concentration of protein was determined using a BCA protein assay reagent kit (Beyotime, China). The proteins (15 μg) were separated by dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Then, the membrane was blocked with 5% skimmed milk for 2 h at room temperature. The PVDF membranes were then incubated overnight at 4°C with the following primary antibodies to: proliferating cell nuclear antigen (PCNA; Cell Signaling Technology, Boston, MA, USA), Ki67 (Santa Cruz Biotechnology, CA, USA), (p-) Raf (Cell Signaling Technology, Boston, MA, USA), (p-) MEK (Cell Signaling Technology, Boston, MA, USA), (p-) ERK (Cell Signaling Technology, Boston, MA, USA), VE-cadherin (Cell Signaling Technology, Boston, MA, USA), Frizzed (Abcam Company, Cambridge, UK), β-catenin (Cell Signaling Technology, Boston, MA, USA), MMP-2 (Abcam Company, Cambridge, UK), MMP-9 (Abcam Company, Cambridge, UK) and GAPDH (Cell Signaling Technology, Boston, MA, USA). Followed by being incubated with a horseradish peroxidase (HRP)-labeled secondary antibody (Abcam, Cambridge, MA, USA). The results were visualized with an enhanced chemiluminescence (ECL) detection kit. ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the density of the bands.

Total proteins from PDCs were isolated using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA) containing a protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitor cocktail (Roche), and protein concentration was determined using a Quick Start Bradford Protein Assay (Bio-Rad, Hercules, CA, USA). Aliquots containing 30 μg of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% v/v Tween 20 and probed overnight at 4°C with specific antibodies against the following proteins: p-EGFR, p-RAF, RAF, p-MEK, MEK, p-ERK, ERK, p-Rb1, Rb1, P-AKT, AKT (Cell Signaling Technology, Beverly, MA, USA), and beta actin (Sigma Aldrich). Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Vector, Burlingame, CA, USA) was used as a secondary antibody, and signals were detected by chemiluminescence using ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA) and visualized using LAS-4000 (Fujifilm, Tokyo, Japan).