Bs 1047r

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.