Gw21317

Endothelial cells were homogenized with RIPA (radioimmunoprecipitation assay) buffer containing protease inhibitor cocktails (Sigma–Aldrich, US) on ice. Equal amounts of protein (30 μg) were denatured in SDS and separated in 10% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes in transfer buffer containing 20% methanol. The membranes were blocked with 5% skim milk, incubated with primary antibody overnight at 4 °C and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, Jackson ImmunoResearch, PA) at room temperature for 1 h. β-Actin (1:2000, sc-47778, Santa Cruz) was used as the internal control. The following primary antibodies were used: anti-RGS12 (1:1,000, GW21317, Sigma–Aldrich), anti-p-Tyr (1:1000, Ab10321, Abcam), anti-MYCBP2 (1:1000, ab86078, Abcam), anti-acetylated Tubulin (1:1000, 66200-1-Ig, Proteintech), anti-IκB (1:1000, 10268-1-AP, Proteintech), anti-NFκBp65 (1:1000, 10745-1-AP, Proteintech), and anti-Ubiquitin (1:1000, sc-166553, Santa Cruz).

Synovium tissue or synovial fibroblasts were homogenized with radioimmunoprecipitation assay buffer containing protease inhibitor cocktails (PICs) and phenylmethanesulfonylfluoride (phenylmethylsulfonyl fluoride [PMSF]) (Sigma-Aldrich) on ice. Equal amounts of protein (30 μg) from the different groups were denatured in SDS loading buffer and separated on 10% SDS-PAGE gels. Proteins were transferred to NC membranes in the transfer buffer containing 20% methanol. Membranes were sequentially blocked with 5% skim milk, incubated with the primary antibodies described below overnight (4°C), and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000, Jackson ImmunoResearch) at room temperature for 1 h. Signals were analyzed using an enhanced chemiluminescence western blot system (Bio-Rad Laboratories). β-Actin (1:3000, sc-47778 HRP; Santa Cruz) was used as the internal control. The following antibodies were used: anti-RGS12 (1:1,000; GW21317; Sigma-Aldrich), anti-KIF2A (1:1,000; 13105-1-AP; Proteintech), and anti-MYCBP2 (1:1,000; MABN2397; Sigma-Aldrich).

In brief, protein lysate was extracted from macrophages and synovium by radioimmunoprecipitation assay buffer. Protein extracts were boiled and subjected to SDS-PAGE and then transferred and incubated as previously described (26 (link)). Anti-β-Actin (sc-8432, Santa Cruz, USA) at a dilution of 1:2,000 was used as the internal control. Anti-RGS12 (1:1,000 dilution, GW21317, Sigma-Aldrich, USA) anti-COX2 (1:1,000 dilution, 4842, CST, USA), anti-NF-κB P65 (1:1,000 dilution, 10745-1-AP, Proteintech, USA), and anti-Flag (1:2,000 dilution, F3165, Sigma-Aldrich, USA) were used. Protein bands were visualized on a ChemiDoc™ touch imaging system (Bio-Rad, Hercules, CA, USA) using a Clarity™ Western enhanced chemiluminescence (ECL) detection kit (Bio-Rad, Hercules, CA, USA). The analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).