Ab205606

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).

Western blot analysis was performed according to protocols previously described.⁴⁰ (link) Briefly, cells were washed twice with cold PBS and scraped in RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 0.1% SDS, and 50 mM Tris [pH 8.0]) supplemented with 0.1 mM phenyl methyl sulfonyl fluoride (PMSF). Cell lysates were incubated on ice for 30 min and centrifuged at 15,000 × g, 4°C for 15 min. Proteins in the supernatant were extracted and quantified using bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Cell lysates were loaded with 4× loading dye (Tris-HCl [pH 7.4], 1% SDS, glycerol, dithiothreitol, and bromophenol blue) and subjected to electrophoresis on 8% or 10% SDS-polyacrylamide gels and then transferred onto nitrocellulose membrane (Bio-Rad, Richmond, CA, USA). The membrane was blocked with 5% milk in Tris-buffered saline (TBS) with Tween 20 and incubated with primary antibodies (anti-FLAG [1:1,000] [Abcam, ab205606], anti-GAPDH [1:5,000] [Proteintech, 60004-1]). After washing with TBS supplemented with 0.05% Tween 20 for 30 min, secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, WestGrove, PA, USA) was added to the membrane for visualization.