Sds reagent

FLS which were cocultured with pretreated articular cartilage were homogenized using sodium dodecyl sulfate (SDS) reagent (Beyotime, Shanghai) supplemented with 1% phosphatase and protease inhibitor cocktail (Thermo Fisher, CN) following the manufacturer's instructions. The protein concentration was assessed with BCA assay kit (Biosharp, China). Next, proteins were separated using electrophoresis, transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 5% bovine serum albumin (Biosharp, China), and incubated at 4°C with primary antibodies for 16 hours. Then, the PVDF membrane with proteins was incubated with secondary antibodies. Fluorescent signals from protein were detected by Odyssey image system (Lincoln, NE). Primary antibodies used here included NF-κB (p65), inhibitor of NF-κBα (I-κBα), I-κB kinase β (IKK-β), CD147, and GAPDH were offered by Cell Signaling Technology (CST, 1 : 1000 dilution). Secondary antibodies used in this study included Anti-mouse IgG (H+L) (DyLight™ 680 Conjugate) and Anti-rabbit IgG (H+L) (DyLight™ 680 Conjugate) were offered by Cell Signaling Technology (CST, 1 : 30000 dilution).

Total proteins were extracted with sodium dodecyl sulfate (SDS) reagent (Beyotime, Shanghai), and eBlot L1 Protein Transfer System (GenScript Corporation, China) was used for protein electrophoresis. SDS-PAGE gel electrophoresis, protein transfer, and immunohybridization were performed to determine changes in expression levels of target genes (Table 3). Finally, the target bands were scanned by a fluorescence scanning imager, and the data were quantitatively analyzed.