Cox 2

Primary human OA chondrocytes were plated in 12-well plates, stimulated with oleocanthal 1-5 µM 12 hours and then incubated with LPS (250 ng/mL) during 24 hours or 30 minutes.
Proteins were extracted using a lysis buffer with a commercial protease inhibitor cocktail (Thermo Fisher). SDS-PAGE and blotting procedure were carried on as previously described [13] . Immunoblots were incubated with the appropriate antibody (iNOS, IkB, p-ERK-1/2 and NF-kB p65 from Cell Signaling MA, USA; COX-2 from DAKO, Denmark; MMP13 from Santa Cruz Biotechnology, USA; ERK1/2 and p-38 from Upstate, p-p38 from Millipore; Lamin B1 from Boster, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore Massachusetts, USA) using horseradish peroxidase-labelled secondary antibody. To confirm equal loading in each sample, the membranes were stripped in glycine buffer at pH 2 and re-blotted with an anti-GAPDH antibody (Sigma). The images were captured and analysed with an EC3 imaging system (UVP). Data obtained were further validated by densitometric analysis using Image J software.