Western blot analysis of NF-κB activation

The tuning of NF-κB signaling was further investigated by western blot analysis on both chondrocytes (2 × 10⁵) and synoviocytes (6 × 10⁴) plated in 24 well—plates in CTR (IL-1β stimulated) and sEV (IL-1β + sEV) conditions at both 4 and 15 h since sEV delivery. At the time of collection the medium was removed, the cells were recovered with a scraper using a small volume of cold PBS with the addition of inhibitors of phophatases and proteases. Then the cells were gently centrifuged and lysed with 20 µl of RIPA buffer. The samples were subsequently loaded, run and transferred to PVDF membranes as detailed previously.

Western blot was carried out with the following antibodies: Phospho-NF-κB p65(Ser536) (rabbit monoclonal antibody, clone 93H1, used at 1:1000, CELL SIGNALLING TECHNOLOGY #3033), and β-actin (mouse monoclonal, clone AC-74, used at 0.8 µg/ml SIGMA-ALDRICH # A2228) that served as loading control. Appropriate anti species HRP conjugated secondary antibodies were from JACKSON IMMUNORESEARCH.