Col11a1

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with 1 × protease inhibitor cocktail (Complete Mini, Roche), 0.2 mM sodium orthovanadate, 20 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride (SIGMA); 25 μg of protein lysate was loaded onto 8% SDS PAGE gels. Immunoblots were performed with antibodies against ITG11A1 (Abnova, 1:700), COL11A1 (Abcam 1:1,000), MMP13 (Thermo Scientific, 1:500), INHBA (Abcam, 1:500), SULF1 (Abcam, 1:500), EPYC (SIGMA, 1:2,000), TNFSF4 (BioLegend, 1:300) and ERK2 (Santa Cruz Biotechnology, 1:2,000) overnight at 4°C.

Tissue collected from mice were fixed in buffered formalin (Fisher Scientific, Hampton, NH, USA) and processed in the tissue core facility at Mayo Clinic, AZ. IHC staining was carried out using 5-µm tissue sections according to standard procedures. Briefly, after antigen retrieval with EDTA buffer (pH 8.0, Newcomer Supply, Middleton, WI, USA), samples were blocked and followed by primary antibody incubation at 4 °C for overnight. A secondary antibody (Cell signaling, Danvers, MA, USA) was applied for 30 min at room temperature. Chromogenic detection of protein expression was determined in the presence of diaminobenzidine (DAB, Agilent, Santa Clara, CA, USA) and scanned by a Desktop scanner (Objective Imaging, Kansasville, WI, USA). Primary antibodies purchased from Thermo (p-SMAD2, COL11A1) and Abcam (VCAN).