Protein Quantification and Analysis in Cartilage

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).