Ab58425

The protein was extracted from the heart tissues using Radio Immuno Precipitation Assay Buffer (RIPA) buffer containing Phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor and quantitated by Bradford method using spectramax M2 (Molecular Devices, Sunnyvale, CA, USA). The protein samples were prepared in SDS sample buffer (2% SDS, 10 mM dithiothreitol, 60 mM Tris‐HCl pH 6.8, bromophenol blue 0.1%) and loaded on 10% PAGE. The samples were electro‐transferred to PVDF membrane and probed with appropriate primary and secondary Horse Radish Peroxidase (HRP) conjugated antibodies. The membrane was developed with the Western blotting detection system ECL (GE Healthcare, Piscataway, NJ, USA) and imaging was done using the gel documentation system ChemiDoc XRS system (Bio‐Rad, Richmond, CA, USA). After stripping, the membranes were reprobed with anti‐GAPDH antibody (Millipore, Billerica, MA, USA) as control. Image Lab densitometry software (Bio‐Rad) was used to analyze the data and normalizing the GAPDH. The description of antibodies is: (i) TIMP4‐ ~26 kD (Abcam, Cambridge, MA, USA) (ab58425), (ii) MMP9 ~92 kD Abcam (ab38898), (iii) DNMT1 ~100 kD Abcam (ab13537), (iv) histone 3 acetylation at lysine 9 (H₃K9Ac) Abcam (ab10812) molecular wt ~18 kD.

The cartilage tissue and chondrocytewere exposed to RIPA lysis buffer (Solarbio) to extract total protein, followed by quantification using bicinchoninic acid (BCA) detection kit (Shanghai UCHEM Inc., China). Afterwards, the separated proteins were transferred onto PVDF membranes (Zhejiang Lianshuo Biotechnology Co., Ltd., China). After blocking with 5% non-fat milk for 2 h, the membranes were incubated overnight at 4℃ with primary antibody, followed by incubated with anti-rabbit IgG (ab14708, 1:5000, Abcam) or anti-mouse IgG (ab3420, 1:5000, Abcam) at room temperature for 60 min. Finally, bands were visualized on gel imaging analysis system (Bio-Rad) using ECL chemiluminescence kit (Beyotime, P0018AS) and quantified using an Image J software (National Institutes of Health, Bethesda, Maryland, USA). The primary antibodies were as follows: anti-WTAP (ab195380, 1:1000, Abcam), anti-ADAMTS5 (ab41037, 1:250, Abcam), anti-MMP13 (ab39012, 1:3000, Abcam), anti-Collagen II (ab34712, 1:1000, Abcam), anti-Aggrecan (ab3778, 1:750, Abcam), anti-TIMP4 (ab58425, 1:750, Abcam), and anti-GADPH (ab8245, 1:1000, Abcam).