Ripa buffer 1

Western blot analyses were performed following standard procedures. After 10 days of treatment with BC (1% w/v) and CS (1% w/v), chondrocytes were lysed in RIPA buffer (1×) (Cell Signaling Technology). Protein concentration was determined using the Bradford method [Bradford, 1976] and 60 µg intracellular proteins were loaded and resolved using 8% SDS–PAGE. The separated proteins were then transferred to nitrocellulose membrane (Amersham). The membrane was blocked in 5% milk, Tris‐buffered saline and 0.05% Tween‐20. Primary antibodies to detect type II collagen and type I collagen (Abcam) were used at 1:250 dilutions. Immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase‐conjugated secondary antibody (Santacruz Biotechnology; 1:5000 dilutions) and reacted with an ECL system (Chemicon‐Millipore). Protein levels were normalized with respect to the signal obtained with anti actin polyclonal antibody (Santacruz Biotechnology; 1:500 dilutions). The semi‐quantitative analysis of protein levels was carried out by the Gel Doc 2000 UV System and the Gel Doc EZ Imager, using quantity one software (Bio‐Rad Laboratories).

The cells’ intracellular total protein content was extracted in the radioimmunoprecipitation assay (RIPA buffer) (1×) (Cell Signaling Technology, USA). Protein concentration was determined using the Bradford method (Biorad Laboratories, Milan, Italy) and 5 μg of proteins was separated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes (Millipore, Bedford, MA), and blocked with 5% skimmed milk in Tris-buffered saline with Tween 20 (TBST). The membranes were incubated with primary antibodies to detect COMP-2 (Abcam, Cambridge, UK) and NF-κB (Santa Cruz Biotechnology, CA, USA) both used at 1:500 dilutions and incubated overnight at 4 °C. After washing with TBST, immunolabeling bands were detected by the chemiluminescence detection system using corresponding horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, CAUSA), diluted 1:10,000 for 1 h at room temperature and reacted with an ECL system (Merck Millipore, Darmstadt Germany). Protein levels were normalized with respect to the signal obtained with an anti-actin antibody 1:500 dilution (Santa Cruz Biotechnology, CA, USA). The semi-quantitative analyses of protein levels were carried out using the Gel Doc 2000 UV System (Biorad laboratories, Milan, Italy) according to the manufacturer’s protocols and previously reported [32 , 33 (link)].