Protein Extraction and Analysis from BMSCs and Cartilage

RIPA buffer (Beyotime, Shanghai, China) was utilized for extracting total proteins from BMSCs and murine cartilage tissues. Quantification of proteins were achieved using a Bicinchoninic acid Protein Assay Kit (Beyotime). Protein samples (20 μg) were resolved with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Invitrogen) [29 (link)]. After blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against Map3k2 (ab33918, 1:10000, Abcam), Aggrecan (13880-1-AP, 1:1000, Proteintech, Chicago, USA), collagen II (Col2, ab34712, 1:1000, Abcam), collagen X (Col10, ab182563, 1:1000, Abcam), matrix metalloproteinase 13 (MMP13, ab39012, 1:3000, Abcam), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS, ab41037, 1:250) and β-actin (ab6276, 1:5000, Abcam) overnight at 4°C. After that, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Abcam) at room temperature for 2 hours. Eventually, protein signals were visualized using an enhanced chemiluminescence kit (Cwbiotech, Beijing, China) with an Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA).