Bca protein concentration determination kit

Dried nymphs of PA were obtained from Sichuan Good Doctor Pharmaceutical Group Co., Ltd. Coomassie brilliant blue fast staining solution was obtained from Beyotime Technology Co., Ltd (Shanghai, China). DL-Dithiothreitol and iodoacetamide were obtained from Adamas Pharmaceuticals, Inc. RIPA lysis solution, BCA protein concentration determination kit, 5x reduced protein loading buffer, sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) gel preparation kit and protein marker were all obtained from Servicebio Technology Co., Ltd (Wuhan, China). Phosphate buffer (powder, pH 7.2–7.4) was acquired from Livning Biotechnology Co., Ltd (Beijing, China). Dulbecco’s modified Eagle medium (DMEM) and penicillin-streptomycin (PS) were acquired from Gibco. Fetal bovine serum (FBS) was acquired from Zhejiang Tianhang Biotechnology Co., Ltd. Dilinoleyl-Dil (Cell membrane orange‒red fluorescent probe), DAPI staining solution and YF®488-Phalloidin were all acquired from US Everbright Inc. Streptozotocin (STZ) was acquired from Yuanye (Beijing) Biotechnology Co., Ltd. Ultrapure water was made in the laboratory.

Western blot analysis was performed between groups. Total proteins were extracted from cell samples. BCA protein concentration determination kit (Servicebio, Wuhan, China) was used to determine the total protein concentration. The proteins were separated by 10% SDS-PAGE (Servicebio, Wuhan, China) and transferred to cellulose nitrate membrane. The membrane was sealed in 5% degreased dry milk in 37°C TBS buffer (Servicebio, Wuhan, China) for 1 h and incubated overnight with primary antibodies (MPO antibodies, Abcam, Europe) at 4°C. Then, the membranes were washed three times in TBST and incubated with secondary antibodies at room temperature for 30 min. A scanner (EPSON V300, Japan) was used to sweep membrane and upload band data. Adobe PhotoShop (Adobe, USA) software was used to process the color, and Alpha Innotech (Alpha Innotech, USA) software was used to analyze the gray values of band. Relative expression level of protein = (gray value of the target protein band)/(gray value of the GAPHD protein band).