Anti histone h2b

Lung tissue was lysed in Pierce™ IP Lysis Buffer (ThermoFisher Scientific). The lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected and the protein concentration in the supernatant was determined by BCA Protein Assay Kit (Bio-Rad Laboratories). For the immunoblot, 40 µg of the total protein was resolved in SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween-20 for 30 min at room temperature. The membrane was incubated with primary antibodies overnight at 4 °C followed by incubation with secondary antibodies. The membrane was then developed using ECL Western Blot Substrate (ThermoFisher Scientific). The following primary antibodies were used: anti-NLRP3 (1:1000, AG-20B-0014-C100), anti-Caspase-1(p20) (1:1000, AG-20B-0042-C100), anti-Asc (1:500, AG-25B-0006-C100) from Adipogen Life Sciences, Anti-p65 (8242, 1:1000), anti-P-p65-Ser536 (3033, 1:500) from Cell Signaling Technology Inc., and anti-β-actin (1:5000, A5441, Sigma-Aldrich). For the immunoblot analysis of BALF samples, equal volumes of BALF from all groups were loaded. The following primary antibodies were used: anti-Histone H2B (1:500, ab52484, Abcam), anti-CitH3 (1:500, ab5103, Abcam),

NETs from PMA-stimulated HC and BD neutrophils with equal DNA quantity were digested with 20 U/ml DNaseI (Sigma, USA) for 30 min at 37°C and stopped with 5 mM EDTA. Proteins were precipitated with cold acetone at −20°C overnight and pelleted at 14,000×g for 10 min at 4°C. The pellets were resuspended in lysis buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM HEPES and protease inhibitors (Beyotime, China). Proteins were loaded with SDS loading buffer, denatured at 95°C for 5 min, and stored at −80°C. NETs proteins were loaded onto 4%–20% polyacrylamide gels, and the gels were transferred to PVDF membranes for 1 h at 200 mA. Membranes were blocked with QuickBlock blocking buffer (Beyotime, China) and were incubated with anti-Histone H1, anti-Histone H2A, anti-Histone H2B, anti-Histone H3, anti-Histone H4, anti-NE, anti-S100A8 (rabbit, Abcam, UK) antibody at 4°C overnight. HRP-conjugated goat anti-rabbit antibodies (Abcam, UK) were incubated for 1 h. Blots were developed with chemiluminescence and detected by Tanon 5200 (China). Protein levels were quantified by Image J software and measured with absolute optical density values, given no reference control protein available.