Ab38473

After cells treated with/without 100 ng/ml rhVEGF, 100 nM apatinib or 100 ng/ml rhVEGF + 100 nM apatinib, cells were lysed using lysis buffer (Cell Signaling Technology, Danvers, USA) to extract total protein. Protein lysates were separated by 10% SDS-PAGE, followed by transfer to nitrocellulose membranes. The membrane was then blocked with 5% milk diluted in PBS at room temperature for 1 h, followed by incubated with 1:1000 VEGFR2 antibody (ab10972, Abcam, Cambridge, MA, USA),1:5000 p-VEGFR2 (ab38473, Abcam), 1:2000 p-MEK (2338, CST), 1:1000 MEK (4694, CST), 1:2000 p-ERK1/2 (4370, CST), 1:1000 ERK (4695, CST), 1:2000 slug (ab51772, Abcam), 1:3000 Snail (ab53519, Abcam), 1:2500 MMP9 (ab38898, Abcam), 1:1500 P-AKT (ab81283, Abcam), 1:1500 AKT (ab179463, Abcam) and 1:5000 GAPDH antibody (ab8245, Abcam) overnight at 4 °C separately. Once primary antibodies were washed, membrane was incubated with goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Sangon Biotech, Shanghai, China). Protein bands were detected by incubating the membrane with Western Bright enhanced chemiluminescence working solution (Advansta, Menlo Park, CA, USA). The film (Kodak XBT-1, Carestream, Xiamen, China) was scanned with Bio-rad Gel Doc XR+ (BIO-RAD, Shanghai, China).

Cell total protein was extracted, then resolved on 10% SDS-PAGE, and transferred onto PVDF (polyvinylidene fluoride) membranes. Non-specific binding sites were blocked by 5% non-fat milk for 1 h at room temperature. Subsequently, the membranes cut and probed with corresponding primary antibodies at 4°C overnight, followed by incubation with the secondary antibodies for 2 hours at room temperature. Primary antibodies information is as follows: anti-VEGFR2 (phospho Y951) (ab38473, Abcam), anti-VEGFR2 (ab39638, Abcam), anti-VEGFA (ab46154, Abcam), anti-SP1 (9389S, Cell Signaling Technology, CST for short), anti-β-Tubulin (2128S, CST).