Vegfa sc 7269

Cells were harvested and proteins were extracted using cell lysis buffer supplemented with 0.3% phenylmethylsulfonyl fluoride and proteinase and phosphatase inhibitors. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking in 5% skimmed milk powder for 2 hours at room temperature, the samples were incubated overnight at 4°C with primary antibodies, including ERK1/2 (#9102), p-ERK1/2 (#9101), JNK1/2 (#9252), p-JNK (#9251), p38 MAPK (#9212), and p-p38 MAPK (#9211), which were obtained from Cell Signaling Technology (Beverly, MA, USA), as well as VEGF-a (sc-7269) and Tubulin (sc-8035), which were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). After washing three times with TBST (Tris-buffered saline supplemented with 0.1% Tween-20), the samples were incubated for 1 hour at room temperature with a secondary antibody. Bound antibodies were detected using the enhanced chemiluminescent substrate (ECL, Pierce, Rockford, IL, USA). The bands were assessed by densitometry with Image J software (National Institutes of Health).