Am063

Western blotting assay was performed to analyse the protein expression of senescence markers (p16 and p53) and p38 MAPK activity. Briefly, after total protein was extracted with the RIPA lysis solution (Beyotime) and protein concentration was measured with the BCA kit (Beyotime), protein samples were subjected to SDS-PAGE system and transferred to a PVDF membrane. After blocking with 5% bovine serum albumin, the PVDF membrane was incubated with primary antibodies (β-actin: Proteitech, Rosemont, IL, USA; 60008-1-Ig; p16: Novus, St. Louis, MO, USA; NBP2-37740; p53: Proteintch, 10442-1-AP; p38 MAPK: AM065, Beyotime; phosphor-p38 MAPK: AM063, Beyotime) at 4 °C overnight and corresponding secondary antibodies (ZSGB-BIO, Beijing, China, diluted 1:2000) at 37 °C for 2 hours. Protein bands were developed with the SuperSignal West Pico Trial Kit (Thermo Fisher Science). Protein expression was analysed with Image J software (National Institutes of Health, Bethesda, MD, USA).

Proteins extracted from isolated neutrophils were used for Western blot analysis. Proteins were separated by 12.5% SDS/PAGE electrophoresis and transferred to PVDF membranes, then the membranes were blocked with 5% BSA at room temperature for 2 h and then incubated overnight at 4 °C with the corresponding primary antibodies: p47^phox (1:800; sc-14015, Santa Cruz Biotechnology), p-p47^phoxSer 304 (1:500, bs-1047R, BioSS, Woburn, MA, USA), p-p47^phoxSer 345 (1:1000, 118391, Sigma-Aldrich, St. Louis, MO, USA), TRAF6 (1:1000, ab40675, Abcam, Cambridge, UK), MyD88(1:500, bs-1047R, Bioss Biotech, Woburn, MA, USA), p38 MAPK (1:1000, AM065, Beyotime Institute of Biotechnology, Beijing, China), p-p38 MAPK (1:1000, AM063, Beyotime Institute of Biotechnology, Beijing, China) and β-actin (1:5000, AP0060, Bioworld, Atlanta, GA, USA). After three washes with TBST, the membranes were incubated with horseradish-peroxidase-conjugated secondary antibody for 2 h. Immunoreactive bands were detected by a chemiluminescence system (ECL Plus; Amersham, Arlington Heights, IL, USA) and analyzed by Quantity One analysis software (Bio-Rad Laboratories Inc., Hercules, CA, USA). β-Actin was used as the internal control.