Anti choline acetyltransferase antibody

DMAB-anabaseine dihydrochloride and PHA568487 were from Tocris Biosciences (Minneapolis, MN, USA); (−)-Nicotine hemisulfate salt and LPS E. coli 0111:B4 were from Sigma (St Louis, MO, USA). Anti-mouse Ly6C APC, anti-mouse CD11b PE, anti-mouse Gr1 FITC and PE, anti-mouse CD4 FITC and PE, anti-mouse CD16/CD32 antibodies and IgG isotype controls were from eBioscience (San Diego, CA, USA). For immunofluorescence, anti-mouse CD11b and Gr1 antibodies were from BD Biosciences (San Jose, CA, USA). Phospho-Akt1 (Ser473) (D7F10) XP rabbit mAb (Akt1 Specific) and PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit were from Cell Signaling (Danvers, MA, USA); AChRα7 antibody (H-302) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse CCR2 APC-conjugated antibody and mouse CXCL2 and TNF-α ELISA Kit were from R&D Systems (Minneapolis, MN, USA). Anti-Choline acetyltransferase antibody was from Abcam (Cambridge, MA, USA). E. coli K1 (serotype) strain, isolated from patients with biliary infection, was kindly provided by Dr Thomas Martin (University of Washington, USA) [69 (link)].

Lung single cells and neutrophils from mice in different treatment groups were collected and lysed with pre-cooling RIPA lysis buffer (Sangon Biotech, Shanghai, China). The lysates were centrifuged at 12,000 g for 20 min at 4℃. The supernatants were collected and the protein concentrations were determined by BCA assay (Beyotime, China). Protein samples were fractionated on 12% Tris-glycine gels, followed by proteins transfer onto a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat dry milk and then probed with diluted primary antibodies against the following mouse proteins, including anti-choline acetyltransferase antibody (Abcam, Cambridge, UK), anti-vesicular acetylcholine transporter (VAChT) antibody (Abcam, Cambridge, UK), anti-PADI4/PAD4 antibody (Abcam, Cambridge, UK). Goat anti-Rabbit IgG-HRP antibody (Abcam, Cambridge, UK) was used as secondary antibody. Antibody binding was visualized with a chemiluminescent substrate on Azure Biosystems (USA).