Anti p 38

The procession of protein extraction in this study can be referred to the article (Luo et al., 2021 (link)). 20 µg per sample of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and deposited onto a polyvinylidene fluoride (PVDF) membrane after the concentration of proteins was determined. The membranes were blocked with 5% milk for 2 h before incubation with the primary antibody at 4°C overnight, which is as follow, Anti-p-38 (Affinity Biosciences, OH, United States), Anti-p-p-38 (Affinity Biosciences, OH, United States), β-Tubulin (Affinity Biosciences, OH, United States). After response with the primary antibody, the blotted PVDF membrane was treated with horseradish peroxidase (HRP) conjugated secondary antibod (Boster Biological Technology Co. Ltd.). The ECL chemiluminescence western blot detection technique was performed with a gel imaging system and the band intensity were analyzed via ImageJ.

Western blotting was performed as described previously [54 (link)]. Briefly, cells and liver tissue samples were collected and lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Solarbio, Beijing, China). Protein concentration was measured using a BCA protein assay kit (LABLEAD, Beijing, China). Proteins were separated by 10% SDS-PAGE gel at 115 V for 1.2 h, then were transferred to a PVDF membrane at 200 mA for 1 h. The membranes were blocked with 5% milk in TBST for 1 h and incubated overnight at 4 °C with primary antibodies. Primary antibodies included anti-Smad4, anti-α-SMA, anti-GAPDH, anti-E-cadherin, anti-ID1, anti-CTGF, anti-p65, anti-p-p65, anti-p38, and anti-p-p38 (Affinity Biosciences, Cincinnati, OH, USA). Followed by HRP-conjugated goat, anti-mouse and goat anti-rabbit IgG (Solarbio, Beijing, China) were used as secondary antibodies. Protein bands were scanned using a Clinx Science Instrument and quantified with Image J software.