Np 40 lysis buffer

Stomach tissues were immersed in NP40 lysis buffer (Solarbio, China) supplemented with protease and phosphatase inhibitors, and sonicated on ice. The homogenates were centrifuged at 15,000× g for 15 min, and the supernatants were aspirated. The protein concentration was assessed using bicinchoninic acid protein assay kit (Beyotime, China), and equal amounts of protein per sample were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 10% gel. The protein bands were transferred onto nitrocellulose filter membranes, blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight with anticleaved caspase-1 (Asp296; CST, Cat. no. 67314), anticaspase-1 (Abcam, Cat. no. ab138483), anti-IL-1β (3A6; CST, Cat. no. 12242), anticleaved-IL-1β (Asp117; CST, Cat. no. 52718), antinuclear factor kappa B (anti-NF-κB) p65 (Abcam, Cat. no. ab32536), and anti-NLRP3 (Abcam, Cat. no. ab210491) primary antibodies at 4°C. The membranes were then probed with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature and visualized using chemiluminescent reagents (Millipore, USA). β-Actin was used as the internal control.

NP-40 lysis buffer (Solarbio, China), PMSF(Solarbio, China) ,and Protease Inhibitor Cocktail(Solarbio, China)were used to extract proteins. Total proteins were transferred to PDVF membranes (GE, America) from 10% SDS–PAGE after determining the protein content (BCA Protein Colorimetric Assay Kit, Elabscience, China). Membranes were incubated in primary antibodies α-SMA (1:1000), Smad2 (1: 2000), Smad3 (1: 1000), phospho-Smad2 (1: 5000), phospho-Smad3 (1: 2000), and p38MAPK (1: 500) at 4 °C overnight. After washing, membranes were incubated with secondary antibodies (Rabbit anti-Goat IgG–HRP, abisin, China at room temperature for 2 h. Chemiluminescence was performed with Thermo ECL (elabscience, China and visualization was done with a Tanon gel imager. We used the Image J software to quantify using β-actin as an internal standard. This experiment was repeated three times to obtain average values.

When the HeLa cells reached the 80–90% confluence, they were seeded and cultured for 12 hours. At the 12^th hour, 20 μM or 50 μM flavonoids (Chengdu Biopurify, China) were added into the well of cell cultured plates and cultured for another 12 hours. At the 24^th hour, the cells were lysed with NP40 lysis buffer (Solarbio, China), and the total protein was prepared and boiled for degeneration. 30 μg total protein was loaded into the SDS-PAGE and run for 2 hours. After 2 hours, the protein samples in gels were transferred to the PVDF membrane (Millipore, USA), blocked with 5% skim milk, and then were incubated with the primary antibody of TGFβ, mTOR, p70S6K, 4EBP1, p-mTOR, p-p70S6K, p-4EBP1, E-cadherin, Snail, FAK, and p-FAK (Proteinteck, China) for immunoreaction, then washed, incubated with secondary antibody, and visualized using an enhanced chemiluminescence system (Proteinteck, China).