Anti nlpr3

Spinal cord tissues of sham mice (n = 6), CCI mice (n = 6), and CCI mice infected with Lenti-miR-223 (n = 6) or Lenti-vector (n = 6) were buffered with RIPA. The solution is lysed, and the total protein is extracted. The separation of proteins was achieved by 10% SDS-PAGE, and then, the separated proteins were transferred to PVDF membranes. Incubate the membrane with 5% milk at room temperature for 1–1.5 hours and then incubate with the primary antibody at 4°C overnight. The primary antibodies are anti-NLPR3, anticaspase-1 cleavage, anti-ASC, anti-IL-1β, anti-IL-18, and anti-β-actin (both from Abcam). The blot was washed again with Tris buffered saline/Tween 20 (TBST) 3 times and then incubated with a secondary antibody diluted 1 : 5000 at room temperature for 1 hour. The blot was washed 3 times with TBST again and then developed by enhanced chemiluminescence. The band intensity was quantified using UN-SCAN-IT gel analysis software, and the protein expression was normalized to the expression of β-actin.

Treated cells were lysed in RIPA containing protease inhibitor (PMSF, 1 : 100) on ice for 30 minutes. The cell lysate was then centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was then transferred to new centrifuge tubes. The protein concentrations were determined using a BCA kit (Beyotime Biotech, Shanghai, China); equal amounts (40 μg) of protein samples were separated using SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk powder in TBST for 2 h. The membranes were probed overnight at 4°C with anti-NLPR3 (1 : 1000, Abcam, UK), anti-ASC (1 : 1000, Proteintech, IL, USA), anti-Caspase1 (1 : 1000, Proteintech, IL, USA), and anti-IL-1β antibodies (1 : 1000, Proteintech, IL, USA). Further, the membranes were incubated with HRP-conjugated secondary antibodies(1 : 2000, Proteintech, IL, USA) for 2 h at room temperature. The antibody binding was detected using the enhanced chemiluminescence (ECL) detection kit (Beyotime Biotech, Shanghai, China).