Pvdf membrane

The synovium tissue and THP-1 cells were collected and lysed in a RIPA buffer (Beyotime, China). The total protein concentration was measured using a BCA protein assay kit (Beyotime, China). Samples containing ~50 mg protein was separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by the transference to polyvinylidene fluoride (PVDF) membranes (Millipore Corporation, MA, USA). Subsequently, PVDF membranes were blocked with 5% (w/v) non-fat milk in TBST buffer for 2 h at room temperature and treated with corresponding primary antibodies (1:500 to 1:1000) overnight at 4˚C. The membranes were washed three times with Tris buffer saline-Tween20 (TBST), followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (1:1000 to 1:2000) for 2 h. Finally, protein bands were visualized with an enhanced chemiluminescence (ECL) system (Keygen Biotech, China) and scanned with a Chemiluminescence imaging system (Gel Catcher 2850, China). The relative optical densities of bands were analyzed with a ChemiScope analysis program.

According to the standard protocol, 80 μg total protein samples were prepared and determined by BCA assay kit (KeyGEN). Then, 20 μg total proteins were separated by SDS-polyacrylamide gel, transferred to a PVDF membrane (KeyGEN), and blocked with 5% BSA in Tris-buffered saline containing 0.5% Tween 20, followed by incubation with primary (polyclonal rabbit-anti-rat SOD2 antibody, ZSGBBIO, China, 1 : 1000) and secondary antibody (goat-anti-rabbit antibody, ZSGBBIO, China, 1 : 8000). β-Actin (primary antibody, 1 : 200; secondary antibody, 1 : 6000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as an internal control. Protein bands were visualized using chemiluminescence reagent (KeyGEN).

The EPA16 PAHs standard reagent, dimethyl sulfoxide (DMSO), ethylene diamine tetraacetic acid (EDTA), and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, USA). The n-hexane and dichloromethane were of high-performance liquid chromatography (HPLC) grade. Triton X-100, and Tris-HCl were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). The propidium iodide (PI) cell-cycle kit, Annexin V-FITC apoptosis detection kit, protein extraction kit, SDS-PAGE, PVDF membrane, and antibodies (primary and secondary) were provided by KeyGen Biotech (Nanjing, China).