P mek1 2

For Western blot analysis, cells were lysed with RIP buffer (Boster) supplemented with RNase inhibitor and phosphatase inhibitor after cell transfection. Protein concentration was identified using a BCA kit (Invitrogen). The lysate sample was separated on a 10% SDS-PAGE gel and blotted onto blotted onto PVDF membranes. The membrane was incubated with primary antibody overnight at 4 °C, including EGFR (1:2000, Abcam), P-EGFR (1:2000, Abcam), KRAS (1:2000, Abcam), BRAF (1:2000, Abcam), MEK1/2 (1:2000, Abcam), P-MEK1/2 (1:1000, Proteintech), ERK (1:2000, Abcam), P-ERK (1:2000, Abcam), MAP3K1 (1:700, Proteintech), P-MAP3K1 (1:1000, Proteintech), β-actin (1:700, Proteintech), incubate for 2 hours at room temperature with anti-rabbit secondary antibody. Finally, protein band detection was performed using a Chemiluminescent Reagent (ECL) kit (Beyotime Biotechnology).

Western blotting was used to detect the expression of proteins involved in apoptosis, angiogenesis and the MEK/ERK signalling pathway at 24 h after administration. Total protein was extracted using protein extraction kit, according to the manufacturer’s instructions and the protein concentration were measured with BCA method. Protein samples (40 μg) were subjected to 10% SDS-polyacrylamide gels electrophoresis (SDS-PAGE) to separate. Then transferred to poly-vinylidnene fluoride (PVDF, Millipore) membranes and blocked in PBST solution with 5% non-fat milk. The membranes were incubated in primary antibodies against brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), Bax, Bcl-2, VEGF, FGF2, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2 (1:2000 dilution, Proteintech, USA) at 4 °C overnight. Secondary antibodies: HRP-conjugated anti-mouse or anti-rabbit IgG (1:5000, Proteintech, USA) were incubated 1 h at 37 °C. Finally, the enhanced chemiluminescent reagent (Thermo Fisher, USA) was added in the membranes and band intensity signals were observed. GAPDH was used as an internal reference, and the optical densities of protein bands were analysed by Image J software to represent the relative expression of target protein.