NF-κB Signaling Modulation in Chondrocytes and Synoviocytes

The tuning of NF-κB signaling was further investigated by western blot analysis on both chondrocytes (100,000) and synoviocytes (60,000) plated in 24-well plates in CTR and sEVs_IL-1 conditions at both 4 and 15 hours since delivery. At the time of collection, the medium was removed, and the cells were recovered with a scraper using a small volume of cold PBS with the addition of inhibitors of phosphatases and proteases. Then, the cells were gently centrifuged and lysed with 20 μl of RIPA buffer. The samples were subsequently loaded, run on the acrylamide gels, and transferred to PVDF membranes as detailed previously [5 (link)].
Western blot was carried out with the following antibodies, Phospho-NF-κB p65(Ser536) (rabbit monoclonal antibody, clone 93H1, used at 1 : 1,000, Cell signaling Technology #3033) and β-actin (mouse monoclonal, clone AC-74, used at 0.8 μg/ml Sigma # A2228) that served as loading control. Appropriate anti-species HRP conjugated secondary antibodies were from Jackson laboratories.