Df13758

Total proteins were extracted from cardiac tissues and H9c2 cells by radioimmunoprecipitation assay lysis buffer (Beyotime) and measured through a bicinchoninic acid protein assay kit (Beyotime). Protein samples (20 μg) were isolated by 10% SDS-PAGE (Beyotime) and transferred into polyvinylidene fluoride (PVDF; Beyotime) membranes. PVDF membranes were then treated with 5% skimmed milk/TBST for 1 h. Subsequently, the membranes were treated with primary antibodies overnight at 4 °C and secondary antibody for another 2 h at ambient temperature. The primary antibodies were as follows: anti-interleukin (IL)-1β (ab254360; 1:1000, Abcam), anti-nucleotide-binding oligomerization domain (NLRP3; ab263899; 1:1000, Abcam), anti-GSDMD-N (DF13758; 1:1000, Affinity), anti-caspase-1 (ab286125; 1 μg/mL, Abcam), anti-ASC (ab180799; 1:1000, Abcam), anti-transforming growth factor (TGF-β1; ab215715; 1:1000, Abcam), and anti-GAPDH (ab181602; 1:1000, Abcam). GAPDH was used as an internal reference. Finally, protein bands were visualized with an electrochemical luminescence on a Tanon 5200 machine (Tanon, Shanghai, China). Protein band intensities were detected with the Image J software (NIH, MD, USA).

The protein levels in cartilage tissues and cells were determined by referring to methods in previous literature [19 (link)]. Briefly, the protein lysates of brain tissues or cell samples were prepared with a radio-immunoprecipitation assay lysis buffer (P1003B, Beyotime, Shanghai, China) and 1% phenylmethylsulfonyl fluoride. Bicinchoninic acid (BCA) kits (Beyotime) were adopted for protein quantification. Subsequently, the proteins (15–50 μg) were separated with 4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes with a pore size of 0.45 μm or 0.22 μm. Next, the membranes were blocked with 5% skim milk for 1 h and cultured with the primary antibodies gasdermin D (GSDMD)-N (dilution ratio of 1:1000, DF13758, Affinity Biosciences, USA), CTSB (dilution ratio of 1:1000, ab214428, Abcam), NLRP3 (dilution ratio of 1:1000, ab263899, Abcam), and GAPDH (dilution ratio of 1:10,000, ab181602, Abcam) at 4°C overnight. Afterward, the membranes were cultured with the secondary antibody anti-rabbit IgG (dilution ratio of 1:1000, ab205718, Abcam) for 1 h, and then visualized using an enhanced chemiluminescence reagent (#34,080, Thermo Fisher Scientific Inc., Waltham, MA, USA). The protein blotting was analyzed using the ImageQuant LAS 4000 (General Electric Company, Schenectady, NY, USA).

H9c2 cells (5 × 10⁵ cells/well) were added into 12-well plates. After fixing in 3% paraformaldehyde for 10 min, cells were washed with PBS (Beyotime) and permeabilized by 1% Triton X-100 (Solarbio, Beijing, China) for 5 min. To determine cell pyroptosis, H9c2 cells were stained with the PI solution for 15 min before being stained with 4ʹ,6-diamidino-2-phenylindole (DAPI; Beyotime) for 5 min at room temperature in dark. Cells were observed under a fluorescence microscope (Olympus).
For detection of expression on gasdermin D (GSDMD-N) and caspase-1, H9c2 cells were further sealed with 3% BSA solution (Solarbio) for 30 min. Next, cells were incubated with primary antibodies anti-N terminal of GSDMD-N (DF13758; 1:100, Affinity, TX, USA) and anti-caspase-1 (ab286125; 1:100, Abcam, Cambridge, UK) at 4 °C overnight and treated with secondary antibody FITC goat anti-rabbit IgG (H + L) (AS011; 1:500, ABclonal Technology, MA, USA) for a further 30 min at room temperature. Nucleus was stained by DAPI (Beyotime). The images were captured under a confocal microscope (Zeiss Microscopy, Jena, Germany).