Ab131441

Neurons grown on 6-well plates and subjected to treatment described above were lysed in RIPA lysis buffer (Thermo Scientific). Protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific) according to the manufacturer’s instructions. Equal amounts of each protein sample (total protein extract, 30 μg) were separated by electrophoresis in 4–15% SDS-polyacrylamide gels (Mini-PROTEAN TGX precast gel, Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes. After being blocked, membranes were incubated overnight at 4°C with primary antibodies against phosphorylated VEGFR-2 (Tyr 996; sc-16629-R, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA), total forms of VEGFR-2 (ab131441, 1:300, Abcam), and β-actin (4967, 1:500, Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:10000, Cell Signaling Technology) for 1 h at room temperature. The blots were developed in enhanced chemiluminescence reagents (Thermo Scientific) and exposed to X-ray film. Signals were quantified with Quantity One software 4.62 (Bio-Rad).