Anti gsdmd

The total protein was extracted from the liver tissues using a Total Protein Extraction kit (Beyotime, Shanghai, China) and quantified using a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein per sample (30 μg) were separated on a 10% SDS–PAGE and electroblotted onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk for 2 h, the blots were incubated overnight with anti-VEGF (1:2000; Wanleibio, Shenyang, Liaoning, China), anti-Cyclin D1 (1:1000; Wanleibio, Shenyang, Liaoning, China), anti-NLRP3 (1:2000; Bioss, Beijing, China), anti-Wnt2 (1:1000; Abcam, Waltham, MA, USA), anti-β-catenin (1:10,000; Abcam, Waltham, MA, USA), anti-P-P65 (1:500; Santa, Dallas, TX, USA), anti-P65 (1:3000; Proteintech, Wuhan, Hubei, China), anti-caspase-1 (1:500; Santa, Dallas, TX, USA), anti-ASC (1:500; Santa, Dallas, TX, USA), anti-GSDMD (1:2000; Affinity, Liyang, Jiangsu, China), and anti-β-tubulin (1:1000; Wanleibio, Shenyang, Liaoning, China) primary antibodies at 4 ℃. The blots were washed with TBST and incubated with HRP-conjugated anti-IgG for 2 h. The positive bands were detected using an enhanced ECL reagent (Meilunbio, Dalian, Liaoning, China) on an AI600 System (GE Healthcare, Pollards Wood, UK). The relative protein expression was quantified using ImageJ software and normalized against the β-tubulin control.

Paraffin sections of rat lung tissue were obtained and rinsed with 0.01 M PBS three times for 5 min each. Then, the tissue was blocked with 10% normal goat serum at 37 °C for 45 min. Suctioned excess liquid and anti-GSDMD (dilution 1:100, Affinity, USA) were added. The sections were incubated at 37 °C for 1 h and then placed in a wet box in a refrigerator at 4 °C overnight. The sections were washed with 0.01 M PBS three times for 5 min each. Goat anti-rabbit IgG-FITC (1:200) was added in the dark and incubated at 37 °C for 45 min. The secondary antibody solution was aspirated and discarded in the dark, and DAPI staining solution (2.5 mg/mL) was added and incubated at room temperature for 20 min. The sections were rinsed with 0.01 M PBS six times for 5 min each in the dark. The sections were then mounted using a fluorescence quencher in the dark and observed under a fluorescence microscope with the appropriate excitation wavelength, and images were acquired to record the experimental results. The fluorescence intensity was assessed using a Zeiss Axioskop 2 (Carl Zeiss MicroImaging, Inc., Weimar, Germany).

Immunostaining of knee joint sections was performed with specific antibodies against target proteins according to a previously published protocol with some modifications (Wang et al. 2019 (link)). Sections were incubated with anti-NLRP3 (1:100), anti-ASC (1:100), anti-caspase-1 (Wanleibio, 1:100), anti-GSDMD (Affinity Biosciences, 1:100), and anti-IL-1β (1:100) antibodies overnight at 4 °C. On the following day, these tissue sections were incubated with a polymer-HRP detection system (PV9001, ZSGB-BIO) and visualized with a diaminobenzidine (DAB) peroxidase substrate kit (ZLI-9017, ZSGB-BIO). Each section was evaluated under a microscope (DMi8, LEICA) at 20 × magnification. Histological analyses were performed in a blinded manner by two independent observers. Image-ProPlus6 software 6.0 (Media Cybernetics, Inc.) was used to analyze the average integrated optical density (IOD) of three randomly selected areas of the acquired images.