Ecl reagent

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).

The lysates of protein samples were collected by using RIPA lysis and extraction buffer (ThermoFisher, 89900), separated by SDS‐PAGE in polyacrylamide gels, and transferred to nitrocellulose membranes. Subsequently, membranes were washed with TBST (50 mM TRIS + 150 mM sodium chloride + 0.1% Tween 20, pH 7.4) and blocked using 5% nonfat milk solution in TBST at least 1 h at room temperature. Membranes were then incubated with primary antibodies: ADAM10 (1:1000, Proteintech, 25900‐1‐AP), ADAM17 (1:1000, Abcam, b57484), IL‐6R (1:1000, Proteintech, 23457‐1‐AP), IL‐6R alpha (1:1000, R&D, MAB227), gp130 (1:1000, Proteintech, 67766‐1‐Ig), JAK2 (1:1000, Proteintech, 17670‐1‐AP), SPON1 (1:1000, Abcam, ab14271), STAT3 (1:1000, Proteintech, 60199‐1‐Ig), mIL‐6R (1:1000, MAB227, R&D), sIL‐6R (1:1000, HCA257, bio‐Rad), P‐JAK (1:1000, CST, 66245), P‐STAT3 (1:1000, CST, 9145S), β‐actin (1:1000, Abcam, ab8227). Molecular weight‐specific clipped bands were incubated with specific primary antibodies at 4°C overnight and were then incubated with HRP‐conjugated secondary antibodies (goat anti‐mouse, 1:10,000, Jackson ImmunoResearch, 115‐035‐003; goat anti‐rabbit, 1:10,000 Jackson ImmunoResearch, 111‐035‐003; goat anti‐chicken, 1:10,000, Abcam) for 1 h at room temperature. The bands were visualized with ECL reagents (ShareBio).

Total proteins were extracted and resolved by SDS-PAGE, transferred to nitrocellulose membranes, blocked by 5% skim milk in 1× TBST buffer, and incubated with the primary antibody at 4 °C overnight. The following primary antibodies were used: anti-GAPDH (Cell Signaling Technology, cat#2118 L, 1:5000), anti-AKT (Cell Signaling Technology, cat#4685 s, 1:2000), anti-phospho-AKT (Cell Signaling Technology, cat#4060 s, 1:2000) anti-CREB (Proteintech, cat#12208-1AP, 1:1000), anti-phospho-CREB (Cell Signaling Technology, cat#9198 S, 1:1000), anti-estrogen receptor alpha (Abcam, cat#ab32063, 1:1000). HRP-conjugated secondary anti-rabbit IgG (Santa Cruz, cat#sc-2357, 1:10,000) was next incubated for 1 h at room temperature. Signals were visualized with ECL Reagent (ShareBio, cat#SBWB012) using GelDoc XR (Bio-Rad).