Af3022

Total protein was extracted from injured spinal cord tissue stored at − 80 ℃ by using radioimmunoprecipitation assay (RIPA) lysis and extraction buffer that included a protease inhibitor cocktail. The concentration of protein was determined by the BCA method. Equal amounts of protein (30–50 µg) from each sample were separated using 4–20% SurePAGE™ Gels (Genscript) and transferred to nitrocellulose (NC) membranes (EMD Millipore Corp). The membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated overnight at 4 °C with the following specific primary antibodies: rabbit anti-iNOS (18,985–1-AP, Proteintech, 1:1000), rabbit anti-C3 (ab200999, Abcam, 1:1000), rabbit anti-GFAP (SAB4300647, Sigma, 1:1000), rabbit anti-JAK2 (3230, Cell Signaling Technology, 1:1000), rabbit anti-pJAK2 (AF3022, Affinity, 1:1000), rabbit anti-STAT3 (12,640, Cell Signaling Technology, 1:1000), rabbit anti-pSTAT3 (ab76315, Abcam, 1:1000), rabbit anti-Lcn2 (ab63929, Abcam, 1:1000), and anti-β-actin (66,009–1-Ig, Proteintech, 1:1000). After incubation with the corresponding secondary antibodies (1:2000) for 1 h at room temperature, the membranes were scanned with ECL-Plus Reagent (Millipore) and observed under an Amersham Imager 600 (General Electric). The band intensity was analyzed by using ImageJ software (NIH).

Protein was isolated from spinal cord tissue or cultured cells by homogenizing the samples using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher). The protein concentration was determined by the BCA assay, and 20–30 μg of protein from each sample was separated by SDS-PAGE (10 or 12%) and transferred onto NC membranes. After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with the following specific primary antibodies overnight at 4°C: C3 (ab200999, Abcam, 1:1000), S100a10 (PA5-95505, Invitrogen, 1:1000), NF-κB (8242, Cell Signaling Technology, 1:1000), pNF-κB (3033, Cell Signaling Technology, 1:1000), Notch1 (3608, Cell Signaling Technology, 1:1000), JAK2 (3230, Cell Signaling Technology, 1:1000), pJAK2 (AF3022, Affinity, 1:1000), STAT3 (12640, Cell Signaling Technology, 1:1000), pSTAT3 (ab76315, Abcam, 1:1000), PI3K (60225-1-Ig, Proteintech, 1:1000), pPI3K (AF3241, Affinity, 1:1000), Akt (4691, Cell Signaling Technology, 1:1000), pAkt (4060, Cell Signaling Technology, 1:1000), and β-actin (66009-1-Ig, Proteintech, 1:1000). After washing with TBST, the membranes were incubated with corresponding secondary antibodies for 1 h and then developed with ECL-Plus Reagent (Millipore), and the bands were visualized using an Amersham Imager 600 (General Electric).