Bca protein concentration assay

The expression levels of GPR120, IL-6, IL-8, and SOX9 were assessed via western blot analysis. Briefly, proteins were extracted from ATDC5 chondrocytes using cell lysis buffer (Cell Signaling Technologies, USA) supplemented with protease inhibitor cocktail. Following centrifugation at 10000 × g, the supernatant protein was collected from the samples. A BCA protein concentration assay was used to assess the protein concentrations in accordance with the manufacturer’s manual (Sigma-Aldrich, USA). Then, 20 μg protein was loaded onto an 8-12% SDS-PAGE gel for electrophoresis and transferred onto a PVDF membrane (Bio-Rad, USA). To block the nonspecific binding sites, the samples were incubated with 5% non-fat milk in TBST, followed by primary antibodies overnight at 4 °C, and HRP-conjugated secondary antibody at room temperature (RT) for 1 h. The protein bands were then visualized via ECL chemiluminescence (Thermo Scientific, USA). The resulting optical density was quantified using Image J software (NIH, USA). The following antibodies were used in this study: GPR120 (1:1000, #ab230869, Abcam, USA); type II collagen (1: 500, #MAB8887, Chemicon, USA); aggrecan (1: 500, #ab3778, Abcam, USA); SOX-9 (1:1000, #ab59252, Abcam, USA); β-actin (1: 10000, #ab8227, Abcam, USA); NF-κB p65 (1:1000, #ab16502, Abcam, USA); Lamin B1 (1: 5000, ab65986, Abcam, USA).