Anti species hrp conjugated secondary antibodies

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.

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.

Stable transduced chondrocytes in both untreated and IL-1β treated conditions were processed for western blot analysis, exploiting SNAP-ID 2.0 device (Merck-Millipore) essentially as described in Minguzzi et al.^{28 (link)}, loading the same cell equivalents for each lane (150,000 or 200,000 cells, according to the different experiment). Primary antibodies against the antigens were as follows: IKKα (mouse monoclonal, clone B78-1, used at 0.5 µg/ml, BD Pharmingen # 556532), IKKβ (polyclonal rabbit anti-human IKKβ, used 1:1000, Cell Signaling Technology, #2684), Phospho-NF-κB p65(Ser536) (rabbit monoclonal antibody, clone 93H1, used at 1:1000, Cell Signaling Technology #3033), phospho-histone H3 (Ser10) (mouse monoclonal, clone CMA312, used at 0.6 µg/ml, MERCK # 05-1336). β-actin (mouse monoclonal, clone AC-74, used at 0.8 µg/ml Sigma # A2228) served as loading control. Appropriate anti-species HRP conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories.