Sc 14015

Prosurfactant protein C (proSP-C) is the precursor of surfactant protein C, which is exclusively produced in alveolar type II cells to prevent lung collapse (31 (link)). Trypatse is one of the characteristic markers of mast cell activation (32 (link)). The expression of NADPH oxidase reflects the level of oxidative stress, and p47^phox and gp91^phox are subunits of NADPH oxidase (33 (link)). Intercellular adhesion molecule-1 (ICAM-1, CD54) is an important early marker of immune activation and response (34 (link)). Western blot analyses were performed, as previously described (24 (link)). The primary antibodies used in the present study were as follows: Anti-ICAM-1 mouse monoclonal antibody (1:500; sc-8439; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), antitryptase rabbit polyclonal antibody (1:500; sc-32889; Santa Cruz Biotechnology, Inc.), anti-proSP-C polyclonal rabbit antibody (1:1000; AB3786; EMD Millipore, Billerica, MA, USA), anti-p47^phox polyclonal rabbit antibody (1:1,000; sc-14015; Santa Cruz Biotechnology, Inc.) and anti-gp91^phox polyclonal rabbit antibody (1:1,000; sc-20782; Santa Cruz Biotechnology, Inc.). The images were analyzed used ImageJ software, version 1.41 (National Institutes of Health, Bethesda, MA, 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.

Rats were anesthetized and transcardially perfused with PBS, followed by 0.1 M PBS containing 4% PFA (pH = 7.4). Perfusion-fixed brains were postfixed in paraformaldehyde overnight, followed by dehydration in 40% sucrose. Coronal brain sections (10 µm thick) were cut on a cryostat (CM1950; Leica Microsystems, Wetzlar, Germany), and the sections were blocked for 2 h in 5% bovine serum albumin in PBS with 0.3% Triton X-100. The sections were then incubated overnight at 4 °C in 3% bovine serum albumin in 0.1% Triton X-100/PBS with the following primary antibodies: anti-p47^phox antibody (1:200; sc-14015)and anti-MPO antibody (1:100; sc-34159) (both were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being rinsed three times with PBS, sections were incubated for 2 h in fluorochrome conjugated secondary antibody. After being rinsed with PBS, the sections were examined under a laser scanning confocal microscope (Carl Zeiss LSM 700, Oberkochen, Germany).