Hrp conjugated secondary antibody

The chondrocytes were preincubated with PA at concentrations of 0, 2.5, 5, or 10 μM for 24 h and then exposed with D-gal (5 mg/ml) for 24 h. Subsequently, the chondrocytes were lysed by radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China) containing a mixture of protease and phosphatase inhibitors. Protein samples were subjected to SDS-PAGE and transferred to PVDF membranes (Bio-Rad, USA). The membranes were blocked by 5% skim milk for 1 h and incubated overnight at 4°C with the appropriate primary antibodies, respectively. On the following day, after washing with TBST for 30 min, the membranes were incubated with HRP-conjugated secondary antibodies (1 : 5000, Abclonal, China) for 1 h at room temperature. The enhanced chemiluminescence method was conducted to observe the bands, and ImageJ software was applied for quantitative analysis.

The heart sample was homogenized using metal beads in RIPA buffer with protease inhibitor (Beyotimem, Shanghai, China) and PMSF (Beyotime, Shanghai, China). The concentration of total protein was determined by the BCA (Boster Biological Technology, Wuhan, China) method. A 5× loading buffer (Beyotime, Shanghai, China) was added to the sample at the ratio of 4:1 and boiled for 5 min at 95 °C. Then, 30 μg protein of each sample was run on a 12% SDS-PAGE gel (Epizyme Biomedical Technology, Shanghai, China), transferred to PVDF membranes (Boster Biological Technology, Wuhan, China) and then blocked with 5% non-fat milk for 60 min. The membranes were incubated with primary antibodies against Gal-3 (ABclonal, Wuhan, China) or GAPDH (ABclonal, Wuhan, China) at 4 °C overnight, and then membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (ABclonal, Wuhan, China) for 1 h. The blots were developed using the ECL chemiluminescence kit (Boster Biological Technology, Wuhan, China). The levels of target proteins were normalized to GAPDH.

Stimulated NP cells were collected and then lysed with RIPA lysis buffer (CWBIO, Beijing, China) for total protein, or with an extraction kit for cytoplasmic and nuclear protein (CWBIO). After being centrifuged and collected, the supernatant containing proteins was then quantified using a BCA assay kit (CWBIO). Afterwards, the proteins were separated by SDS-PAGE and electro-transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). After being blocked with 5% bovine serum albumin in TBST for 1 h, the membranes were then incubated at 4°C overnight with anti-CBX4 (1:1000; Abcam), anti-MMP3 (1:1000; Abcam), anti-MMP13 (1:1000; Abcam), anti-ADAMTS5 (1:1000; Abcam), anti-COX2 (1:1000; Abcam), anti-COL2A1 (1:1000; Abcam), anti-P53 (1:1000; Abcam), anti-P21 (1:1000; Abcam), anti-β-actin (1:1000; Abclonal, Wuhan, China), anti-p-p65 (1:1000; Abclonal), anti-p65 (1:1000; Abclonal), or anti-Histone H3 (1:1000; CST, Danvers, USA) antibodies. Afterwards, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:8000; Abclonal). The protein bands were visualized using an Enhanced Chemiluminescence kit (Vazyme, Nanjing, China) and then quantified by Image J (National Institutes of Health, Bethesda, USA).

Renal tissues were lysed in RIPA lysis buffer containing protease inhibitor. Equal amounts of proteins (50 μg) were loaded and separated by 10% SDS-PAGE. The proteins were transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk in Tris Buffered saline Tween (TBST) for 1 h at room temperature and were then incubated with appropriate primary antibodies against GPX4 (1:10000, Abcam), ACSL4 (1:10000, Abcam, UK), MLKL (1:1000, Abgent), p-MLKL (1:1000, Abcam), RIPK3 (1:1000, ABclonal), p-RIPK3 (1:1000, Abcam), GSDMD (1:1000, Santa) and GAPDH (1:5000, ABclonal) at 4 °C overnight. The blots were washed with TBST for 5 min and incubated with HRP-conjugated secondary antibodies (1:5000; ABclonal) for 1 h. After washing with TBST, the blots were visualized by the enhanced chemiluminescence method (ECL, Bio-Rad). The relative intensity of the target band was normalized to the corresponding loading control intensity and quantified by using ImageJ (NIH, USA).