Ripa buffer

In RIPA buffer (Epizyme), Cell samples were harvested and then centrifuged at 12,000 × g and 4°C for 15 min. We collected supernatants and used a BCA Kit (Beyotime) to calculate protein concentrations. And 40–60 μg of each sample was taken, and the SDS‐PAGE electrophoresis separated proteins and transferred them into polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies against PES1 (Proteintech, #13553‐1‐AP) and GAPDH (Proteintech, #60004‐1‐Ig) overnight at 4°C after 2 h, closed with 5% milk powder at room temperature. At room temperature, the signal was incubated with a secondary antibody for 1 h after washing 3 times with TBST. Then, according to the manufacturer's recommendations, the signal was detected by enhanced chemiluminescence.

Proteins were extracted from tissues or cells with RIPA buffer (Epizyme, China). Total protein concentration was determined using a protein concentration detector. Proteins were separated by SDS-PAGE (Epizyme, China), transferred onto PVDF membranes (Millipore, Germany), and blocked with a solution of skim milk powder at a concentration of 5% for two hours. The target proteins were probed with corresponding primary antibodies at 4 °C overnight, and then washed with TBST followed by incubation with an HRP-conjugated secondary antibody at room temperature for one hour. Finally, the bands on membranes were visualized through chemiluminescence, and gray value analysis was performed using ImageJ software (v1.53, NIH, USA). Manufacturers and catalogs of antibodies used were listed in additional file 1: Table S1.

The cell lysate was created by treating the cells with RIPA buffer from Epizyme in Shanghai, China, and the amount of protein was measured using the BCA Protein Quantification Kit. A 30 μg protein sample was subjected to electrophoretic separation on a 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were moved to PVDF membranes. Following incubation with a 5% skim milk solution in TBS-T for four hours at room temperature, primary antibodies against TEAD1 (diluted 1:2000, purchased from Abcam, catalog number ab133533, UK) and HIF-1α (diluted 1:2000, acquired from Abcam, catalog number ab79546, UK) were left overnight at 4°C. To ensure equal loading of protein samples, an anti-β-actin antibody (diluted at 1:5000, sourced from Abcam, catalog number ab6275, United Kingdom) was employed. After washing the membranes three times with TBS-T for 15 minutes each, they were then incubated with secondary antibodies (diluted at 1:10,000; Westang) conjugated to horseradish peroxidase for 45 minutes at room temperature. Protein bands were visualized using a developing solution (Vazyme, Nanjing, China) and exposed to X-ray film.

Protein extraction and immunoblot analyses were performed as described below. Cells were lysed in RIPA buffer (EpiZyme, Shanghai, China). The lysates were centrifuged and the supernatants were collected. Cell lysate (20 μg) was separated by SDS–polyacrylamide gel electrophoresis, transferred onto PVDF membranes. After blocking, membranes were incubated with a primary antibody: rabbit monoclonal against SERPINE2, KRT81, KRT7, DKK1, β-Catenin, WNT3A, NF-kB p65, VEGFC, TNFR1, GAPDH (diluted 1:1,000; ABclonal, China), and mouse monoclonal against N-Cadherin, MMP-9 (diluted 1:1,000; Cell Signaling Technology, USA) for overnight at 4°C. After repeated washing, the membranes were incubated with horseradish-peroxidaseconjugated anti-mouse or anti-rabbit secondary antibody (diluted 1:10,000; ABclonal, Shanghai, China). The bands were visualized using the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech). Each experiment was performed in triplicate.

Cells were lysed by using RIPA buffer (Epizyme, China) containing protease inhibitor (AbMole, USA). The total proteins were separated by 10% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore, USA). After blocking with 5% fat-free milk, incubate with primary antibodies against TIMP1 (A00561-1; Boster, China) and β-actin (Beyotime Biotechnology, China), respectively, using a dilution of 1 : 1000 for each antibody overnight at 4°C. After washing with TBST, secondary antibodies were introduced into the membrane for 1 h at room temperature. The interested proteins were visualized using the enhanced chemiluminescence (ECL, Epizyme), and the protein band intensity was analysed using ImageJ software.