Ab53519

RIPA lysis buffer was added into the cell suspension and reacted for 30 min at 4°C. Subsequently, the samples were boiled for 10 min and centrifuged at 12,000 × g for 5 min at room temperature. The supernatants were collected and the quantity of protein loaded per well was adjusted. The protein sample (20 mg) from each lysate was separated by using 10–12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis, transferred onto nitrocellulose membranes. The membranes were blocked using 5% non-fat milk in PBS (pH 7.2) for 2 h at room temperature and incubated with primary antibodies overnight at 4°C, including anti-Snail (1:500; ab53519; Abcam, Cambridge, MA, USA), anti-MMP-2 (1:100; ab135562; Abcam) and GAPDH (1:400; ab8245; Abcam). The membranes were then washed with 1% TBS-Tween and incubated with secondary antibodies for 2 h at room temperature. An ECL chemiluminescent substrate kit was used to detect the reaction and images were analyzed using AlphaEase FC software version 3.0 (ProteinSimple, San Jose CA, USA). The western blot assays were conducted in at least three independent experiments.

Cells (please see below) were lysed by using lysis buffer for western blotting (P00l3, Beyotime) and loaded onto 10% sodium dodecyl sulphate-polyacrylamide gels and were then transferred onto polyvinylidene difluoride membranes (Millipore). The quantification of total protein was performed using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific), and equal amounts of protein (30 μg) were used for analysis. Blots were blocked with 5% milk/TBST and incubated with primary antibodies (Twist1: 1:500, sc-15393, Santa Cruz Biotechnology; Slug: 1:1,000, 6591, Cell Signaling Technology; Snail: 1:1,000, ab53519, Abcam; Vimentin: 1:1,000, 2707-1, Epitomis; EphA2: 1:500, sc-924, Santa Cruz Biotechnology; VE-cadherin: 1:500, ab33168, Abcam; MMP2: 1:500, ab37150, Abcam; E-cadherin: 1:200, SC-8426, Santa Cruz Biotechnology; p-smad2/3: 1:500, sc-11769, Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with secondary antibodies (goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP, 1:2,000; sc-2005 and sc-2030; Santa Cruz Biotechnology) for 2 h at room temperature. Blots were developed using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech). For protein loading analyses, p-actin antibody (1:1,000, P30002, Abmart) or GAPDH (1:2,000, ab9485, Abcam) was used.

Total protein from tissue specimens and cells was extracted by sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 5% 2-β-mercaptoethanol, Sigma-Aldrich, USA). The concentrations of protein were then determined using the BCA protein assay kit (Beyotime, China). Western blot analysis was performed as described 14 (link). Briefly, after being separated in SDS-PAGE gels, proteins were transferred to nitrocellulose membranes (Bio-Rad, USA). The membranes were then incubated with different primary antibodies at 4°C, overnight. Afterwards, respective secondary antibodies were applied at room temperature for 1hour. ECL development solution was manipulated for the visualization of the expression of different protein. The antibodies used are listed as follow: anti-HIF-1α (dilution at 1:1000, A11945, Abclonal, China), anti-FAP (dilution at 1:800, BM5121, Boster, China), anti-E-cadherin (dilution at 1:1000, 3195, CST, USA), anti-Twist (dilution at 1:1000, ab175430, Abcam, England), anti-Snail (dilution at 1:1000, ab53519, Abcam, England).

Proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membrane. After blocking in 5% BSA/PBST for 2 hrs, the PVDF membrane was probed with WTAP (60188-1-Ig, Proteintech, China), E-cadherin (20874-1-AP, Proteintech, China), N-cadherin (22018-1-AP, Proteintech, China), Slug (ab106077, Abcam, USA), Snail (ab53519, Abcam, USA), β-catenin (51067-2-AP, Proteintech, China), GAPDH (60004-AP, Proteintech, China). Protein expression was detected using enhanced chemiluminescence (KeyGen, Nanjing, China).