Pvdf membranes

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.

HMGB1/TLR4/PKR pathway-related proteins were measured by western blot. The BCA kit (71285, NOVAGEN, Darmstadt, Germany) was used to evaluate the protein concentration. We mixed the protein sample (30 μg) with 10% SDS gel buffer at a 1: 1 ratio and then put it in 95°C water for 5 min to degrade the protein.
The samples were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA), and 5% nonfat dry milk was used to block the membrane at 4°C. Then, the PVDF membranes were incubated with primary antibodies against HMGB1 (1: 500, Biorbyt, Cambridge, UK), TLR4 (1: 500, Biorbyt, Cambridge, UK), PKR (1: 2000, Abcam, Cambridge, UK), and GAPDH (1: 1000, Santa Cruz, CA, USA) at 4°C for 12 h. After washing, the membranes were incubated with corresponding secondary antibodies (1: 2000, Santa Cruz, CA, USA) for 2 h at room temperature. The protein was colored for 3–5 min with ECL luminescent substrates (Beyotime, Jiangsu, China) after washing. GAPDH, as an invariant control, was also measure by western blot.