Western Blot Analysis of Spinal Cord Proteins

Western blotting was carried out as previously described [20 (link)]. Proteins were extracted from cells with the protein extraction buffer (Beyotime, Shanghai, China). Spinal cord samples (epicenter ± 0.3 cm) were collected and homogenized with 10 to 15 strokes (3–4 s/stroke) using a homogenizer and plastic pestle with protein extraction buffer. Subsequently, protein concentration was determined using the Bradford method, and equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with the following corresponding primary antibodies overnight at 4 °C followed by blocking with 5% skimmed milk or 5% Bovine Serum Albumin (BSA): anti-β-actin (1:1000), anti-MSR1 (1:1000), anti-cleaved-Caspase-3 (1:1000), anti-Bcl2 (1:1000), anti-Bax (1:1000), anti-IκBα (1:1000), and anti-pIκBα (1:1000). After incubating with species-matched secondary antibodies (1:10000), ECL reagents (Share-bio, Shanghai, China) were used to develop bands and the density of protein was accessed by the ImageJ (National Institutes of Health, Bethesda, MD, USA).