Mouse anti β actin

Protein was extracted from the thoracic aorta of mice and HUVECs with RIPA lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with five percent skimmed milk at room temperature for 2–3 h and then incubated overnight at 4 °C with antibodies. The antibodies used were HDAC11 (1:250, Abcam, ab18973), GSDMD (1:1000, Cell Signaling Technology, #93709), GSDMD-N (1:1000, Cell Signaling Technology, #36425), GSDME (1:500, Proteintech, 13075-1-AP), GSDME-N (1:1000, Abcam, ab222407 and ab222408), TNFR1 (1:500, Wanleibio, Shenyang, China, WL01414), TNFR2 (1:500, Wanleibio, WL02956), pro-caspase-1 (1:1000, Wanleibio, WL02996), cleaved caspase-1 (1:1000, Wanleibio, WL02996a), NLRP3 (1:1000, Wanleibio, WL02635), ASC (1:1000, ImmunoWay Biotechnology Company, YT0365), pro-caspase-3 (1:500, Wanleibio, WL04004), cleaved caspase-3 (1:500, Wanleibio, WL01992), ERG (1:500, Proteintech, 14356-1-AP), and mouse anti-β-actin (1:1000, Cell Signaling Technology, #58169S). Secondary antibody and the enhanced chemiluminescent substrate were used for detection. Relative protein expression levels were semiquantitatively performed using Lane 1D software (Sage Creation Science Co, China).

Cell extracts were prepared by lysis in 1× SDS loading buffer; then, the samples were boiled and subjected to Western blot analysis. Proteins (30 μg) were transferred from gels onto PVDF membranes (Millipore, Billerica, MA, USA) and blocked in 5% skimmed milk for 2 h. The following antibodies were used in the experiment to detect proteins: rabbit anti-SIRT1 (1:1000, Abcam, Cambridge, UK), EGR1 (1:1000, Abcam), USF2 (1:1000, Abcam), NF-κB p65 (acetyl K310) (1:1000, Abcam), TIMP-2 (1:1000, Abcam), MMP-2 (1:500, ProteinTech, Wuhan, China), MMP-9 (1:500, ProteinTech), mouse anti-β-actin (1:2000, Cell Signaling Technologies, Beverly, MA, USA), and NF-κB p65 (1:100, Santa Cruz, Dallas, Texas, USA). The antibodies were incubated with the membranes at room temperature for 1 h and then at 4 °C overnight. HRP-linked anti-rabbit and anti-mouse secondary antibodies were incubated with the membranes at 4 °C for 1 h. All blotting was detected by the enhanced chemiluminescence (ECL) detection method.

Nigericin and LPS were purchased from InvivoGen Biotech Co., Ltd. (San Diego, CA, USA). Mouse anti-FLAG, mouse anti-myc, mouse anti-β-actin, mouse anti-HA, rabbit anti-IL-1β, rabbit anti-NLRP3, rabbit anti-Casp-1, and rabbit anti-ASC monoclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated anti-rabbit antibody or anti-mouse antibody were purchased from ZSGB-BIO, Lnc. The translation inhibitor cycloheximide (CHX) was purchased from APEXBIO (Houston, TX, USA). Lipofectamine 2000 was purchased from invitrogen (Waltham, MA, USA).

Cell pellets were resuspended in 500 μl RIPA buffer (10X RIPA buffer with protease inhibitors). Lysates were incubated on a shaker for 1 h at 4°C, after which they were centrifuged for 30 min at 19,467 × g to remove cell debris. Supernatant protein concentration was assessed using the Bradford assay, 20 μg total protein was resolved using a 10% SDS-PAGE gel at 180 V for 50 min, and then transferred onto PVDF membranes at 22 V overnight. Membranes were blocked in 5% dried milk in phosphate buffered saline (PBS). Primary and HRP-conjugated secondary antibodies were resuspended at the appropriate concentrations in 5% dried milk in PBS. Western blot analysis was performed with rabbit anti-Fibronectin (1:50,000, Sigma, F3648), rabbit anti-Collagen I (1:500, Abcam, ab34710), rabbit anti-dentin matrix protein 1 and rabbit anti-dentin phosphohoryn [1:1000 (Hao et al., 2009 (link))], rabbit anti-osteopontin (1:1000, Abcam, ab8448), and mouse anti-β-actin antibodies (1:1000, Cell Signaling Technologies, cst3700S); and developed using Pierce enhanced chemiluminescence (ECL) Plus western blotting substrate (ThermoFisher, Cat. No. 32106).