Anti histone h2a

All the antibodies used in the study were purchased commercially, including Anti-Flag tag (Sigma, catalog F7425), anti-Myc (HuaBio, catalog EM31105), anti-BAP1 (Santa Cruz Biotechnology, catalog sc-28383 for IP and WB; GST, catalog 78105 for ChIP), anti-ASXL1 (Abcam, catalog ab228009; Abcam, catalog ab221455), anti-FOXK1 (Abcam, catalog ab18196), anti-FOXK2 (Abcam, ab5298), anti-HCF-1 (GST, catalog 50708), anti-H2AK119 mono-ubiquitination (Abcam, catalog ab193203), anti-H3K27me3 (Abcam, catalog ab6002), anti-Histone H2A (Abcam, catalog ab18255), anti-Histone H3 (CST, catalog 9715), anti-TXNIP (Abcam, catalog ab188865), anti-HIF-1α (Abcam, catalog ab216842), anti-STAT3 (CST, catalog 12640), anti-p-STAT3 (CST, catalog 9145), anti-JAK2 (Abcam, catalog ab108596), anti-p-JAK2 (Abcam, catalog ab32101), anti-GAL4 (Santa Cruz Biotechnology, catalog sc-510), anti-ACTIN (Genscript, catalog A00702).

The following primary antibodies were used for Western blotting: anti-GFP (Roche Applied Science, monoclonal and in-house polyclonal antibody raised against GFP(2–238)); anti-ubiquitin FK2 (Enzo) and anti-HA tag (Cell Signaling and Bethyl Laboratories); anti-His tag (Cell Signaling); anti-DCNL1 clone 3D7 (Sigma); anti-β-actin (Cell Signaling); anti-CUL1 (Invitrogen); anti-CUL2 (Invitrogen); anti-CUL4B (Sigma); anti-Elongin-C (BioLegend); anti-RBX1 (ThermoFisher Scientific); anti-CAND1, anti-IκBα, and anti-phospho(S536)–p65 (Cell Signaling); anti-CSN5 (Abcam); anti-Hif1α (R&D Systems); anti-RBX2 (Abcam); anti-CSN3 (Bethyl Lab); anti-CSN7B (Epitomics); anti-CSN8 (Abcam); and anti-histone H2A (Abcam). Polyclonal antibodies against CUL3, CUL4A, CUL5, HHARI, and TRIAD1 have been described previously (28 (link)).

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.