Anti par antibody

3,3′-diaminobenzidine (DAB) staining was performed with quenching of endogenous peroxidase activity with 40% MeOH and 10% H₂O₂ in PBS for 20 min. The sections were incubated with 10% normal goat serum (NGS) in PBS containing 0.1% Triton X-100 for 1 h followed by anti-PAR antibody (1:200; Enzo Life Sciences, Lörrach, Germany; ALX-804-220-P100) incubation for 1 h. Incubation with the biotinylated secondary antibody (1:150, Vector Laboratories Inc., Burlingame, CA, USA; BA-9200; in 5% NGS in PBST) for 1 h was followed by application of Vector ABC-Kit (Vector Laboratories, Burlingame, California, USA; PK-4000) for 1 h. DAB staining solution (0.05 mg/ml NH₄Cl, 200 mg/ml glucose, 0.8 mg/ml nickel ammonium sulphate, 1 mg/ml DAB, 0.1 vol. % glucose oxidase in PB) was applied evenly, incubated for exactly 60 s and immediately rinsed with PB to stop the reaction. The sections were mounted in Aquatex (Merck, Darmstadt, Germany; 1.08562.0050).

Neutrophils were stimulated with LPS for 4 h at 37°C. Neutrophils were co-cultured with or without A549 or LLC cells, as described above. Then, the neutrophils were washed twice in PBS and solubilized at the indicated time points in RIPA lysis buffer with a complete protease inhibitor cocktail. The lysis debris was cleared after centrifugation at 12,000 × g for 10 min. The lysates were then immunoprecipitated with an anti-PARP-1 antibody or an equal amount of normal IgG. Protein A/G Sepharose beads (Santa Cruz Biotechnology, Inc.) were added. After incubation for 2 h at 4°C, the Protein A/G Sepharose beads were centrifuged and washed five times with RIPA lysis buffer. The immunoprecipitants were analyzed by immunoblotting. Antibodies for immunoblotting analysis included anti-ALOX5 and anti-PARP-1 antibodies. For PARylation detection, the immunoprecipitants of anti-ALOX5 antibody were analyzed using an anti-PAR antibody (Enzo Life Sciences, Farmingdale, NY, USA) by western blot. For IP/MS, the immunoprecipitants were analyzed by MS (LTQ Orbitrap Velos; Thermo Scientific).