Western blot
SECONDARY
Total protein was extracted from rat blood samples using RIPA protein lysate (Solarbio, Beijing, China). Protein content was quantified using the BCA Protein Concentration Assay Kit (Solarbio). The proteins were separated by 8% SDS-PAGE and transferred to PVDF membranes, which were closed with 5% skimmed milk at ambient temperature for 2 h. Next, the membranes were subjected to cultivation with primary antibodies against JAK2 (1:500), p-JAK2 (1:1000), STAT3 (1:500), p-STAT3 (1:1000) and β-actin (1:1000) (all from Cell Signaling Technology, Inc., USA) overnight at 4 °C, followed by 1 h-cultivation with horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody at 37 °C. Protein bands were tested using an ultra-sensitive enhanced chemiluminescence kit (Beyotime, Shanghai, China). The grayscale values of each protein band were analyzed using Image-Pro Plus (version 6.0, Media Cybernetics, Inc.).