Ab51772

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).

OSCC cell protein was harvested by ice-cold RIPA lysis solution, and protein concentration was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). The protein was separated by SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane by wet transfer method. Finally, the protein bands were sequentially probed with primary and HRP-conjugated secondary antibodies, after which protein-antibody complex was visualized by enhanced chemiluminescence (ECL) reagent.
Antibodies for GAPDH (ab9485, Abcam, Cambridge, UK), SNAIL (ab216347, Abcam), and SLUG (ab51772, Abcam) were purchased from Abcam. Antibodies for TAB2 (14410-1-AP, Proteintech, Rosemont, USA), PI3K (20584-1-AP, Proteintech), and AKT (60203-2-Ig, Proteintech) were purchased from Proteintech. Antibodies for Caspase-3 (9662s, Cell Signaling Technology, Boston, USA), Caspase-8 (4790S, Cell Signaling Technology), Caspase-9 (9504S, Cell Signaling Technology) were purchased from Cell Signaling Technology.

The total proteins were extracted from HPAEpiCs or lung tissues by using lysis buffer (P0013G, Beyotime, China), and the protein concentration in each extract was determined using a Bradford protein assay kit (ab102535, Abcam, Cambridge, UK. Next, a 20 ug sample of total protein from each extract was separated by SDS-PAGE (10%; Beyotime), and the protein bands were transferred onto PVDF membranes (Millipore, Billerica, MA, USA), which were subsequently incubated overnight at 4°C with the following antibodies: anti-LC3B (1 : 1000, ab51520, Abcam, Cambridge, MA, USA), P62 (1 : 1000, ab91526, Abcam), Beclin-1 (1 : 1000, ab207612, Abcam), SLUG (1 : 1000, ab51772, Abcam), HIF-1α (1 : 500, ab92498, Abcam), E-cadherin (1 : 500, ab40772, Abcam), N-cadherin (1 : 1000, ab202030, Abcam), and GAPDH (1 : 10000, ab8245, Abcam). Next, the membranes were incubated with an HRP goat anti-rabbit/mouse IgG secondary antibody (1 : 20000, ab8245, Abcam); after which, the immunostained protein bands were detected using an enhanced chemiluminescence detection system (Millipore). The integral optical density values of the protein bands were analyzed using Image Pro Plus 6.0 software.

Cells in each group were lysed with RIPA lysate (1 mL RIPA + 10μL PMSF + 10μL aprotinin) for 30 min and operated on ice. Protein concentration was measured by BCA method, and loading buffer was added to denatured protein. Prepare 10% SDS-PAGE and add 20 μg protein sample to each well. Use wet transfer to transfer to PVDF membrane. 5% skimmed milk powder was closed for 2 h. Primary antibody of PAR2 (ab180953, Abcam, Cambridge, MA, USA), Oct4 (ab200834), SOX2 (ab171380), Nanog (ab109250), Snail1 (ab31787), Slug (ab51772)), Twist1 (ab50887)), E-cadherin (ab40772), Vimentin (ab92547) was diluted at 1: 1000 TBST at 4 ℃ overnight. The 1: 5000 diluted secondary antibody was added and incubated for 2 h at room temperature. ECL luminescence kit was developed and analyzed after fixing.