Rats were euthanized by anesthetic over‐dose with isoflurane inhalation. The lumbar spinal cord (L3–5) were collected and homogenized to obtain protein extract for Western blot analysis. Protein extract was centrifuged at 16,000g for 15 min at 4°C.
The equal quantified proteins (50 μg) were denatured and loaded for separation by 10%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to the polyvinylidene fluoride membrane (Millipore). Membranes were blocked with 5% nonfat milk/TBST for 2 h at room temperature and probed with the following primary antibodies: rabbit anti‐IL‐6, anti‐IL‐6Rα, anti‐JAK2, anti‐pJAK2, anti‐STAT3, anti‐pSTAT3, anti‐MIF, anti‐Cox‐2, and anti‐β‐actin (1:1000; Affinity). Subsequently, the membranes were incubated with 1:10,000 horseradish peroxidase‐conjugated goat anti‐rabbit secondary antibody (Affinity) in 5% nonfat milk/TBST for 1 h at room temperature. Finally, proteins were visualized with ECL reagent (Affinity) and analysed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
The equal quantified proteins (50 μg) were denatured and loaded for separation by 10%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to the polyvinylidene fluoride membrane (Millipore). Membranes were blocked with 5% nonfat milk/TBST for 2 h at room temperature and probed with the following primary antibodies: rabbit anti‐IL‐6, anti‐IL‐6Rα, anti‐JAK2, anti‐pJAK2, anti‐STAT3, anti‐pSTAT3, anti‐MIF, anti‐Cox‐2, and anti‐β‐actin (1:1000; Affinity). Subsequently, the membranes were incubated with 1:10,000 horseradish peroxidase‐conjugated goat anti‐rabbit secondary antibody (Affinity) in 5% nonfat milk/TBST for 1 h at room temperature. Finally, proteins were visualized with ECL reagent (Affinity) and analysed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).