Anti pjak2

Samples containing equal protein concentrations were added to 4× protein loading buffer and boiled in a water bath for 5 min. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to wet PVDF membranes. The modified membranes were sealed in 5% milk for 3 h, and then incubated with the respective primary antibodies (anti-STIP1 [1:2000], anti-JAK2 [1:1000], anti-beta-actin[1:5000], Proteintech; anti-pJAK2 [1:1000], Affinity; anti-STAT3 and anti-p-STAT3 [1:1000], Cell Signaling) at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies at room temperature for 50 min. Beta-actin was used as the internal control.

Total tissue protein was extracted from cell lysates (Beyotime, Shanghai, China), and the protein concentration was determined using a bicinchoninic acid (BCA) protein quantification kit (Beyotime). Protein samples were electrophoresed on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk powder at room temperature for 2 h, incubated with appropriately diluted primary antibody at 4 °C overnight, rinsed 3 times, and incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 2 h. The bands were visualized with a fluorescence chemiluminescence imaging system (Clinx Science Instruments, Shanghai, China). Anti-TGF-β1, anti-α-SMA, anti-Fn, anti-GAPDH, anti-JAK2, anti-p-JAK2, anti-STAT3, and anti-p-STAT3 monoclonal antibodies were purchased from Affinity (Jiangsu, China). Anti-TGF-β-R2 antibodies were purchased from Proteintech. Antibody dilutions were purchased from Beyotime. HRP-labeled goat anti-rabbit and goat anti-mouse IgG antibodies were purchased from EarthOx (USA).