Af4012

Radio immunoprecipitation assay (RIPA) lysis buffer (EpiZyme, Shanghai, China) was used to extract total protein from aortic tissues and HUVECs. The protein concentration was measured using the BCA detection method. Subsequently, the equivalent amount of protein was separated by 12.5% SDS‐PAGE, then wet transferred to PVDF membranes (Millipore, Boston, MA, USA) and blocked with 5% non‐fat milk for 1 h at 37°C, followed by incubated overnight at 4°C with the rabbit anti‐NLRP3 antibody (1:1000, ab263899, Abcam, MA, USA), rabbit anti‐cleaved caspase‐1 p20 antibody (1:2000, AF4005, Affinity, OH, USA), rabbit anti‐cleaved‐IL‐1β antibody (1:2000, AF4006, Affinity, OH, USA), rabbit anti‐GSDMD antibody (1:2000, AF4012, Affinity, OH, USA) or anti‐β‐actin antibody (1:1000, #4967, Cell Signaling Technology, USA). β‐actin served as the housekeeping protein control. After washing with TBST three times, the membranes were incubated with the relevant HRP‐conjugated secondary antibody at 37°C for 1 h. The chemiluminescence detection kits (Meilunbio, Dalian, China) were used to visualize protein bands. Finally, ImageJ software was used for densitometric analysis.

The samples added lysis buffer were incubated on ice for 15 min and centrifuged at 12,000 r/min. The supernatant was then gotten. After measuring the protein concentration in the samples via the bicinchoninic acid method, the samples were mixed with sodium dodecyl sulfate loading buffer and boiled for 15 min. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Blocking with 5% skimmed milk solution or 5% bovine serum albumin solution, the membrane was cut into suitable strips and incubated with primary antibodies of MIF (88186, Cell Signaling Tech, Boston, Massachusetts, USA), NLRP3 (DF7438, Affinity Biosciences, Jiangsu, China), ASC (DF6304, Affinity Biosciences, Jiangsu, China), P-p65 (AF2006, Affinity Biosciences, Jiangsu, China), p65 (AF5006, Affinity Biosciences, Jiangsu, China), Caspase-1 (p45, p20) (22915-1-AP, Protein Tech, Wuhan, China), GAPDH (60004-1-Ig, Protein Tech, Wuhan, China), GSDMD and its N-terminal fragment (AF4012, Affinity Biosciences, Jiangsu, China) at 4 °C overnight. The strips of membrane were washed by 1× TBST solution and then incubated secondary antibodies. The bands of proteins were developed by an electrochemiluminescence imaging system (Tanon-5200, Shanghai, China).

The protein from tissue or cultured cell was extracted using RIPA buffer (R0010, Solarbio, China). Lysates were obtained by centrifugation at 4°C with 12000 rpm for 15 min. Protein levels were quantified with a BCA protein assay kit (Beyotime Biotechnology, China). Then, total protein was separated via SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Lot#K5NA8025F, Millipore, USA). Block membranes with 5% skim milk TBS-T for 1.5 h. Incubate them overnight at 4°C with primary antibodies against NLRP3 (1 : 1000, ET1610-93, Huabio), ASC (1 : 1000, DF6304, Affinity), caspase-1 (1 : 1000, ET1608-69, Huabio), IL-1β (1 : 1000, AF5103, Affinity), GSDMD (1 : 1000, AF4012, Affinity), and GAPDH (1 : 1000, 181602, Abcam). The membranes were then washed with TBS-T, followed by incubation with secondary antibodies at room temperature for 1.5 h. After washing 3 times with TBS-T, blot signals were visualized by chemiluminescent kit (Millipore, USA).

The total protein of MCs was extracted with RIPA lysis buffer. The protein samples were subjected to SDS-PAGE (8%–10%) separation and bound to the polyvinylidene difluoride membrane. Then the protein bands were blocked with 5% skim milk and incubated with a primary antibody against NLRP3 (A5652, 1;1,000, ABclonal Technology, Woburn, MA, USA), ASC (bs-6741R, 1:1,000, Bioss, Woburn, MA, USA), caspase-1 (GB11383, 1:1,000, Servicebio, Wuhan, China), TXNIP (14715, 1:1,000, Cell Signaling Technology, Danvers, MA, USA), GSDMD (AF4012, 1:1,000, Affinity Biosciences, Cincinnati, OH, USA), GAPDH (20770-1-AP, 1:1000, Proteintech, Wuhan, China), N-GSDMD (ab215203,1:1000, Abcam, Cambridge, MA, USA), and cleaved-caspase1 (sc-398715, 1:200, Santa Cruz, Dallas, TX, USA) overnight at 4°C. The horseradish peroxidase-labeled secondary antibody and ECL detection kit were used to detect the target proteins. The ratio of the gray value of the target protein to the corresponding GAPDH was the relative expression of the target protein.

Immunofluorescence was performed as per standard protocols. The suitably treated chondrocytes were fixed with 4% PFA for 30 min and permeabilized in 0. 3% Triton X-100 at RT for 30 min. Tissue sections were treated with xylene and alcohol gradient, and heated in citric acid for antigen retrieval. After incubating with 10% serum for 1 h at RT to block non-specific binding, the cells or tissue sections were incubated overnight with primary antibodies against collagen II (1:200, Abcam, ab34712), MMP13 (1:200, Proteintech, 18165-1-AP), NLRP3 (1:200, Affinity, DF7438), GSDMD (1:100, Affinity, AF4012), p-IκBα (1:200, Affinity, AF2002) and p-p65 (1:1000, CST, 3033) at 4 °C. The following day, the cells or tissue sections were incubated with fluorochrome-conjugated rabbit or mouse secondary antibodies (1:300, Invitrogen, A-11008 anti-rabbit or A-11001 anti-mouse), and mounted with DAPI medium (Solarbio). The samples were observed under a fluorescence microscope (Olympus IX73), and the fluorescence intensity of the positively stained cells or tissue area was measured using Image J.