Horseradish peroxidase conjugated goat anti rabbit or goat anti mouse igg secondary antibody

Total protein lysates from synovial tissues and cells were extracted by using the RIPA solution (Beyotime, China) with a cocktail of protease and phosphatase inhibitors (Roche). The total protein concentration of each sample was determined by a BCA Protein Assay kit (Thermo Scientific, USA). Subsequently, 20 μg from cell lysates was separated by 6% or 8% SDS-PAGE gels and transferred to the polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was incubated with primary antibodies at 4 °C overnight. The list of primary antibodies is depicted in supplemental Table S5. After washing, the membrane was further incubated with a horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG secondary antibody (0.4 μg/mL, Abcam, USA) for 2 h at room temperature. Signal intensity was determined by the Supersignal® West Pico Kit (Thermo Scientific) using the enhanced chemiluminescence detection system (EMD Millipore). The band density was measured by the ImageJ software normalized to β-actin.