Co-immunoprecipitation (Co-IP)

Cell lysates were harvested and centrifuged at 14,000 rpm for 20 min to remove the debris after primary joint capsule fibroblast treatment with 2 μg/mL MIF (ProSpec, USA) for 24 h. Protein concentration in the supernatant was quantified using the Protein Assay Kit II (Bio-Rad). For immunoprecipitation analysis, total cell lysates (500 μg) were precleared with protein A plus G-Sepharose (Beyotime) before incubation with specific antibodies at 4 °C, followed by addition of protein A plus G-Sepharose. After several washes, samples were boiled and analyzed by immunoblotting using anti-MIF or anti-CD74 antibody.