Ab39012

Protein extraction was performed by lysing primary MCs and C28/I2 cells in RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease inhibitor (Fudebio, Hangzhou, China). The protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime). Equivalent amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (EMD Millipore). The membranes were blocked and then incubated with primary antibodies for 12 h at 4°C, then incubated with corresponding secondary antibodies (Proteintech, Wuhan, China) for one hour. β‐actin was used as the internal standard, and the relative grey level of proteins was calculated using ImageJ software (NIH, Bethesda, MD, USA). The antibodies used were as follows: anti‐β‐actin antibody (1:2000, Proteintech, 66009‐1‐Ig), anti‐LOX1 antibody (1:1000, Proteintech, 11837‐1‐AP), anti‐Aggrecan antibody (1:1000, Sigma–Aldrich, c8035), anti‐COL2A1 antibody (1:1000, abcam, ab34712), anti‐MMP3 (1:1000, Proteintech, 17873‐1‐AP), anti‐MMP13 (1:1000, abcam, ab39012), anti‐ADAMTS4 (1:1000, Proteintech, 11865‐1AP), anti‐ADAMTS5 (1:1000, abcam, ab41037), anti‐Vimentin (1:1000, Cell Signaling Technology, 5741), anti‐FLAG (1:1000, abcam, ab205606), and anti‐SYVN1 (1:1000, Proteintech, 67488‐1‐Ig).

The knee joints were fixed in 4% paraformaldehyde for 24 h, and decalcified in 10% EDTA (pH 7.4) for 14 days before being embedded in paraffin. The paraffin-embedded tissue was cut into 5-μm-thick sections. The articular sections were treated with pepsin (0.25 mg/ml, Sigma) for antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (for immunohistochemistry). The cells were fixed with 4% paraformaldehyde. The cells and sections were then blocked with 5% bovine serum albumin (BSA, Sigma, USA) and incubated overnight with rabbit-anti Collagen X (Abcam, 1:200, ab58632), rabbit-anti MMP-13 (Abcam, 1:200, ab39012), rabbit-anti RSK-3 (Proteintech, 1:200, 14446-1-AP), rabbit-anti p-RSK-3 (RD systems, 1:200, AF893), rabbit-anti CD44 (Abcam, 1:200, ab157107), mouse-anti CD90 (Abcam, 1:200, ab225), or rabbit-anti Ki67 (Abcam, 1:200, ab15580) diluted in 3% BSA. Finally, the cells and sections were incubated with Alexa Flour 488/546-conjugated donkey anti-rabbit/mouse secondary antibody (Invitrogen, USA) for immunofluorescence or HRP goat anti-rabbit/mouse secondary antibodies (KPL, USA) for immunochemistry. Diaminobenzidine tetrahydrochloride (ZSGB-Bio, China) was used for immunochemical staining. Images were captured under a microscope (Olympus BX51, Japan) and quantitative analysis was conducted in a blinded manner using Image-Pro Plus 6.0 software.