Chondrocyte Protein Profiling via Western Blot

Rat primary chondrocytes were sonicated in radioimmunoprecipitation assay (RIPA) lysis buffer (Gibco, Grand Island, NY, USA). The protein concentration of different groups should be normalized. The protein was separated by electrophoresis and blocked by 5% skim milk after transmembrane. After that, the membranes were incubated overnight with primary antibodies targeting MYD88 (Abcam), matrix metallopeptidase 3 (MMP3, Proteintech), MMP13 (Proteintech), ADAMTS-5 (Abcam), collagen II (Abcam), SOX9 (Proteintech), Aggrecan (Abcam), Bcl-2 (CST), Bax (CST), cleaved caspase-3 (CST), NLR Family Pyrin Domain Containing 3 (NLRP3, Affinity), apoptosis-associated speck-like protein containing a CARD (ASC, CST), cleaved caspase-1 (CST), gasdermin D (GSDMD, Affinity), IL-1β (CST), IL-18 (CST), phosphorylated IκBα (p-IκBα, Affinity), IκBα (Affinity), p-p65 (CST), p65 (CST), and GAPDH (CST) at 4° C. Dilute the primary antibody at 1:1000. After incubation by secondary antibody (1:1000) for 90 minutes, ECL luminescent solution exposed WB bands. We followed our previous methods [19 (link)].