Ecl kit

The total protein was extracted by lysate. A BCA kit was used to quantify protein concentration; then, protein was transferred to the PVDF membrane, and after being resolved by SDS-PAGE (Millipore, Carrigtwohill, Ireland), DRP1 (1:500; Affinity Biosciences, OH, USA), MFN2 (1:1000; Affinity Biosciences, OH, USA), Adiponectin (APN, 1:1000; Zen BioScience, Chengdu, China), AMPK (1:1000; Affinity Biosciences, OH, USA), p-AMPK (1:1000; Affinity Biosciences, OH, USA), SIRT1 (1:2000; Boster, Wuhan, China), PGC-1α (1:1000; Zen BioScience, Chengdu, China), NRF1 (1:1000; Zen BioScience, Chengdu, China), TFAM (1:2000; Affinity Biosciences, Chicago, IL, USA) and β-actin (1:1000; internal control, Boster, Wuhan, China) were incubated together with the membrane in TBST overnight. A TBST wash and 2 h incubation with secondary antibodies followed. Lastly, protein signals were detected with an ECL kit (Boster, Wuhan, China).

RIPA Lysis Buffer (Beyotime, China) containing PMSF (Beyotime, China) and PhosSTOP (Sigma-Aldrich, USA) was used to lyse the cells with indicated treatment. After quantification, the protein samples were separated by SDA-PAGE gel (Beyotime, China). Primary antibodies (Anti-RIPK1, BD Biosciences, USA; Anti-RIPK3, Abcam, USA; p-RIPK3, Abcam, USA; Anti-MLKL, Sigma-Aldrich, USA; Anti-p-MLKL, Sigma-Aldrich, USA; Anti-GAPDH, Beyotime, China) were used at 4 °C overnight. The protein bands were detected by ECL Kit (BOSTER, China).

Chondrocytes were harvested from culture plates to obtain whole cell extracts. Briefly, the chondrocytes were rinsed with PBS, immediately exposed to RIPA Lysis Buffer (Beyotime) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) on ice, followed by centrifugation (12,000×g, 4 °C) for 20 min to clear the lysates. Total protein concentration was measured by bicinchoninic acid (BCA) protein assay (Beyotime). Aliquots containing equal amounts of protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE); protein sample (20 μg) was loaded per lane. Samples were run on 10% gradient polyacrylamide gels (15% polyacrylamide for LC3 detection) and transferred to PVDF membranes (Millipore, USA). After transfer, the membranes were blocked with 5% bovine serum albumin (BSA) and incubated overnight with the specific primary antibodies followed by HRP-conjugated secondary antibodies. Protein detection was visualized with ECL kit (Boster) according to the manufacturer's recommendations. The protein bands were captured by the ChemiDoc XRS gel documentation system (Bio-Rad), and the intensities of bands were quantified by digital image analysis software (Quantity One, version 4.6, Bio-Rad). GAPDH quantification was used as an internal control and served to correct for variations in total protein loading.

Fresh lumbar spinal cord tissue was extracted and put into RIPA lysate (AR0102-100, Boster), adding protease inhibitor PMSF(AR1178, Boster) and phosphatase inhibitor (AR1183, Boster). BCA protein concentration assay kit (AR0146, Boster) was used to determine the protein concentration.
The total proteins from the spinal cord were separated by electrophoresis using 10% SDS–polyacrylamide gel, then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature, then incubated overnight at 4 °C with the following antibodies: Rabbit Anti-SHH Polyclonal Antibody (bs-1544R, Bioss), Rabbit Anti-Gli-1 Polyclonal Antibody (bs-1206R, Bioss), Phospho-AKT (Ser473, Cell Signaling Technology), AKT polyclonal antibody (AP0095, Bioworld) or β-actin (AP0060, Bioworld) antibodies. After washing, the membranes were incubated with Goat anti-Rabbit IgG (BA1054, Boster) for 1 h at room temperature. The blots were visualized by the chemiluminescence-based detection kit (ECL Kit, Boster). Densitometry analysis was performed with a System Gel Doc XR + IMAGE LAB (Bio-Rad, USA) and quantified with NIH Image software (Image J).

The heart apical tissue was lysed for protein extraction. Totally 100 μg of total protein was separated by SDS-PAGE and then electronically transferred onto the PVDF membrane. After blocking with 10% non-fat milk at room temperature for 2 h, the membrane was treated with the primary antibodies against GAPDH (1:10,000 dilution; Abcam, Cambridge, UK), NICD (1:1,000 dilution; Abcam), NF-κB P65 (1:1,000 dilution; Abcam), P-NF-κB P65 (1:1,000 dilution; Abcam), Hes1 (1:1,000 dilution; Abcam), TNF-α (1:1,000 dilution; Abcam), IL-6 (1:1,000 dilution; Affinity Biosciences, China), and caspase-3 (1:2,000 dilution; Abcam), respectively, at 4°C overnight. Then, the membrane was treated with goat anti-rabbit IgG H&L (HRP) (1:20,000, Abcam) at 37°C for 2 h. Protein bands were developed with the ECL kit (Boster, Wuhan, Hubei, China) and analyzed with the Image Lab system. GAPDH was used as an internal reference.