Anti histone h1

The total protein was extracted from 1 × 10⁶ Cos-7 cells. BCA kit was used for protein quantification. 15 μg protein was loaded and run in SDS-PAGE gel. The bands were blocked with 5% skim milk for 1 h at RT. Next the membranes were incubated with the primary antibody diluted in 3% BSA overnight at 4°C. These bands were washed in TBST and incubated with the secondary antibodies for 1 h at RT. Finally, all protein bands were then washed and imaged by chemiluminescence. Grey analysis of bands for agarose gel and western blot were performed by image Lab software. The intensity value was normalized by GAPDH. The primary antibodies were listed as follows: p35/P25: Lot: 2680S, clone number: C64B10, CST; CDK5: Lot: 14145, clone number: D1F7M; CST; Anti-NGF: Lot: ab52918, clone number: EP1320Y, abcam; Anti-Histone H1; #ab4270. The secondary antibodies were listed as follows: Goat anti-mouse IgG secondary antibody: Lot: 3012, Signalway Antibody; Goat anti-Rabbit IgG Secondary Antibody HRP conjugated, Lot: L3012, Signalway Antibody.

Cytoplasmic and Nnuclear extracts were obtained using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, USA) following with the manufacturer's instructions. Total cell lysates were prepared with a detergent lysis buffer. Western blots were carried out as previously reported [36 (link), 37 (link)]. The rabbit anti-ING4 (1:2000, Abcam, USA), anti-MMP-2 (1:1000, Cell Signaling Technology, USA), anti-MMP-9 (1:1000, Cell Signaling Technology, USA), anti-VEGF (1:1000, Abcam, USA), anti-COX-2 (1:5000, Abcam, USA), anti-Sp1 (1:5000, Abcam, USA), anti-histone H3 (1:2000, Abcam, USA), anti-ubiquitin (1:1000, Abcam, USA), anti-cyclinA (1:1000, Cell Signaling Technology, USA), anti-cyclinE (1:1000, Cell Signaling Technology, USA), anti-CDK2 (1:2000, Abcam, USA), anti-p21 (1:1000, Cell Signaling Technology, USA), anti-p27 (1:1000, Cell Signaling Technology, USA), anti-histone H1 (1:2000, Abcam, USA) and anti-p53 (1:1000, Santa Cruz Biotechnology, USA) were used for primary antibody incubation at 4°C overnight. The mouse anti-α-tubulin (1:1000, beyotime institute of biotechnology, China) was used for the protein loading control. Each blot was repeated at least three times. The intensity of the protein bands were analyzed by densitometry after normalization to the corresponding protein controls.

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.

HeLa S3 and HEK293 cells were grown in Dulbecco's modified essential medium supplemented with 10% fetal calf serum. Antibodies used in this study are as follows: Anti-STAT1α/β (sc-346; Santa Cruz Biotechnology), anti-STAT2 (sc-476; Santa Cruz Biotechnology), anti-IRF9 (sc-10793; Santa Cruz Biotechnology), anti-phospho-(Tyr701) STAT1 (#9171; Cell Signaling Technology), anti-phospho-(Tyr689) STAT2 (#07-224; Upstate Biotechnology), anti-phospho-(Ser727) STAT1 (#06-802; Upstate Biotechnology), anti-Pol II (sc-899; Santa Cruz Biotechnology), anti-β-Actin (A5441; SIGMA), anti-Histone H3 (ab1791; Abcam), anti-acetyl-Histone H3 (06-599; Millipore), anti-Histone H1.2 (ab4086; Abcam), anti-Flag (F3165; SIGMA) and anti-TAF-Iα/β (monoclonal antibody KM1725) (34 (link)) antibodies.

Cells were lysed by sonication in ice-cold RIPA buffer containing 1x Protease Inhibitor Cocktail (Roche). Proteins were then separated by SDS-PAGE and transferred to 0.2 µm PVDF membranes (Bio-Rad). Membranes were blocked with Superblock T20 (TBS) blocking buffer (Thermo Fisher Scientific) and probed with primary [anti-Histone H1.2 (Abcam, ab181977, 1:2000 dilution); anti-FLAG M2 (Sigma, F1804, 1:1500 dilution), or anti-beta-actin (Sigma, A2228, 1:5000 dilution)] and secondary [(polyclonal goat anti-rabbit (Dako, P0448, 1:10,000 dilution) or polyclonal goat anti-mouse (Dako, P0447, 1:10,000 dilution)] antibodies or HRP-conjugated p53 antibody (DO1 – sc126, Santa Cruz Biotechnology, 1:1000 dilution). Reactive bands were visualized with Immobilon western chemiluminescent HRP substrate (Millipore) in a luminescence imager (LAS4000, Fujifilm).