Mmp 3

Protein extraction, quantification, and western blot was performed as previously described (19 (link)). The membranes were probed with SLC12A6 (Boster Bio, Pleasanton, CA), MMP-3 (Boster Bio), SSX2IP (Novus Biologicals, Centennial, CO), TRAIL-R4 (Boster Bio), GBP1 (Novus), GUCY1A3 (Novus) or TRPC4 (Boster Bio) primary antibodies. Membrane developing was conducted by enhanced chemiluminescence and autoradiography in an SRX-101A film processor (Konika Minolta, Japan). The ImageLab software was used to quantify the density of bands. All membranes were reprobed with a β-actin monoclonal antibody (Sigma) as a loading control.

Proteins were extracted from synoviocytes and protein concentrations were adjusted using the bicinchoninic acid protein assay kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). Every well in the 10% separating SDS-PAGE was loaded with 20 μL protein. SDS-PAGE was used to separate equal quantities of protein and separated proteins were transferred to nitrocellulose membranes (Fermentas, Thermo Fisher Scientific, Waltham, MA USA). The membranes were treated with PBS containing 5% nonfat dry milk, and incubated with primary antibodies [MMP-3 (1:1,000, Boster, Wuhan, China), MMP-13 (1:1,000, Boster, Wuhan, China), COX-2 (1:1,000, Boster, Wuhan, China) and iNOS (1:1,000, Boster, Wuhan, China)] overnight at 4 °C. After washing, the membranes were treated with horseradish peroxidase-conjugated secondary antibodies, followed by visualization using an enhanced chemiluminescence kit (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). Blots were scanned using a gel imaging system (GelDoc-It 310, UVP Co., Upland, CA, USA) and densitometric analyses were performed using Image Lab 4.1 software (Bio-Rad Laboratories, Hercules, CA, USA).