Rabbit anti human vegf

Proteins were extracted from TECs by using the NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, USA). Equal amount of proteins was loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride filter (PVDF) membranes, and then incubated with antibodies. The membranes were developed with SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Scientific, USA). In brief, samples were boiled in SDS sample buffer for 7 min at 80°C, loaded onto a 10% SDS-PAGE, subjected to electrophoresis for 60 min, and electrophoretically transferred to PVDF membranes. To reduce nonspecific binding, the membrane was blocked with 5% nonfat milk in PBS containing 0.1% Tween 20, incubated overnight at 4°C with primary antibodies in PBS-Tween buffer, washed 3 times with PBS-Tween buffer, and then incubated with a suitable secondary antibody for 60 min at room temperature. The following antibodies were used: β-actin (Cell Signaling Technology Inc., USA), rabbit anti-human VEGF, antibody against phospho-p38 (p-p38) and total p38 (Santa Cruz Biotechnology, Inc., USA), and horseradish peroxidase-conjugated secondary antibody.