Ecl method

Protein samples were prepared using a RIPA lysis buffer. The concentration of the protein was analyzed using a nucleic acid and protein microanalyzer (Molecular Devices, San Jose, CA, USA). SDS–PAGE was performed to separate the proteins, which were then electrotransferred onto a PVDF membrane. The membrane was incubated with antibodies at 4 °C overnight. Next, the membrane was washed and incubated with an HRP-conjugated secondary antibody (Beyotime, BeiJing, China). Finally, signals were detected by the ECL method (Beyotime, BeiJing, China) and analyzed using ImageJ software.

Rabbit monoclonal anti-Glut1 (#12939), rabbit monoclonal anti-LDHA (#3582), rabbit monoclonal anti-PDK1 (#13037), and mouse monoclonal anti-β-actin (#3700) were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). Proteins from cells were extracted using RIPA lysis buffer (Beyotime, China) and separated by SDS/PAGE (10% gel). Subsequently, proteins from gel were transferred on to PVDF membranes. After blocking by 5% nonfat milk for 1 h at room temperature, membranes were probed with primary antibodies at 4°C overnight, followed by incubation with horseradish peroxidase conjugated secondary antibodies (Beyotime, China). After washing completely, membranes were detected by ECL method (Beyotime, China).

After treatment, cells were lysed in RIPA buffer containing 1 mM DTT, 11 μg/ml DNase I and protease inhibitor cocktail (Roche), and incubated on ice for 30 min. Sixty micrograms of protein samples were separated by electrophoresis on a denatured 10% SDS-PAGE gel and blotted onto PVDF membranes (Millipore). Immunoblotting was performed using primary antibody (anti-CHK1 (ab40866, Abcam)); rabbit monoclonal antibody to GAPDH (EPR16891, Abcam) was used as a loading control. The protein was visualized by ECL method (Beyotime Biotechnology, Shanghai, China). The original blots were presented in Supplementary Figs. 1 and 2.

Cartilage tissues were weighed and then lysed by RIPA lysate on the ice for 15 min. Then, samples were centrifuged with 12,000 g/min for 15 min at 4°C. The supernatant was removed. Cultured chondrocytes were washed and lysed by RIPA. BCA protein assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to quantify the protein levels. The protein samples were electrophoresed in SDS-PAGE to separate protein bands. Proteins were transferred from gel onto PVDF membrane, blocked with 5% non-fat dry milk for 2 h. The PVDF membrane was incubated with monoclonal rabbit antibodies specific for MMP-3 (ab52915, Abcam), MMP-13 (MAB13426, Sigma-Aldrich), SOX9 (ab185966, Abcam), Collagen II (ab188570, Abcam), CH25H (sc-293256, SantaCruz), CYP7B1 (ABN182, Sigma-Aldrich) and RORα (ab256799, Abcam) overnight at 4°C. On the next day, membranes were washed and then incubated with second antibody for 2 h. The bands were visualized by ECL method (Beyotime Biotechnology Co., Ltd., Shanghai, China) and the overall gray value of protein bands (average gray value x gray value area) was quantified with Photoshop CS5 software to calculate the target protein relative value (target protein gray value/internal reference overall gray value).