Quantifying Inflammatory Signaling in Chondrocytes

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.