Ab216995

Cells were lysed in cell lysis buffer (Thermo Fisher Scientific, Pittsburgh, USA). The protein concentration in the cell homogenate was determined using a bicinchoninic acid (BCA) assay kit (Beyotime, Shanghai, China). SDS-PAGE separated the proteins (30 μg), which were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Biorbyt, Cambridge, UK). After blocking with 5 % skim milk for 2 h, the membranes were incubated overnight at 4 °C with rabbit antibodies against GBP5 (1:2000, 13,220–1-AP, Proteintech, Wuhan, Hebei, China), IRF1 (1:1000, 8478s, CST, Boston, USA), MMP9 (1:1000, ab228402, Abcam, MA, USA), MMP13 (1:1000, bs10581 R, Bioss, Boston, MA, USA), COL2A1 (1:1000, ab34712, Abcam), aggrecan (1:1000, AF6126, Beyotime), Sox9 (1:1000, BS1597, Bioworld, Nanjing, Jiangsu, China), NLRP3 (1:1000, bs1001 R, Bioss), Caspase1 (1:1000, BS5641, Bioworld), GSDMD (1:1000, ab219800, Abcam), Pro-IL-1β (1:1000, ab216995, Abcam), Pro-Caspase1 (1:1000, ab179515, Abcam), and GAPDH (1:1000, 5174s, CST, Boston, USA). Subsequently, the membranes were incubated with secondary antibodies (1:20,000, BS13278, Bioworld) at room temperature for 1.5 h. After washing the membranes in TBST, chemiluminescent signals were detected using a Bio-Rad Molecular Imager ChemiDocTM XRS + system (Bio-Rad, Hercules, CA, USA), with GAPDH serving as the loading control.

Proteins were extracted and quantified using radio immunoprecipitation assay (RIPA) buffer (Biovision, American) and a protein concentration detection kit (absin, Shanghai, China). The prepared protein was separated by polyacrylamide-sodium dodecyl sulfate (SDS) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Switzerland). Next, the membranes were incubated overnight at 4 ℃ with polyclonal antibodies: cleaved-caspase-1 (#4199T, 1:1,000, Cell Signaling Technology, USA), NOD-like receptor family pyrin domain containing 3 (NLRP3) (ab263899, 1:1,000, Abcam, UK), apoptosis-associated speck-like protein containing a CARD (ASC) (#13833S, 1:1,000, Cell Signaling Technology, USA), gasdermin D (GSDMD)-N (ab215203, 1:1,000, Abcam, UK), interleukin (IL)-1β (ab216995, 1:1,000, Abcam, UK), IL-18 (ab243091, 1:1,000, Abcam, UK), and β-actin (ab8226, 1:1,000, Abcam, UK). After washing with Tris-HCl buffered saline with 0.1% (v/v) Tween 20 (TBST), the membranes were incubated with a secondary antibody, and protein expression intensity was determined by enhanced chemiluminescent (ECL) kit (Beyotime, Beijing, China).

Total cellular proteins were prepared using ice-cold RIPA buffer containing 1% halt protease inhibitor (Beyotime). Proteins (50 µg/sample) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk followed by incubation with primary anti-IL-6 (ab214429), anti-TNF-α (ab183218), anti-IL-1β (ab216995), anti-IL-18 (ab207324), anti-NLRP3 (ab270449), anti-ASC (ab155970), anti-GSDMD-N (ab215203), anti-β-actin (ab8226) (1:1,000, Abcam, USA), and anti-cleaved caspase-1 (#89332) (1:1,000, Cell Signaling Technology, USA) antibodies at 4 ℃ overnight. The membranes were washed and incubated with anti-mouse (ab97040) and anti-rabbit (ab7090) HRP secondary antibodies (1:2,000, Abcam), and the protein bands were detected with enhanced chemiluminescence (Bio-Rad).