Anti histone h4

Different cells were harvested after treatment and proteins were extracted to detect the expression by Western blotting as previously described with minor modifications [46 (link)]. Equal amounts of proteins were size fractionated by 9 to 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Anti-SIRT6 (2590 S, Cell Signaling Techology, Danvers, MA, USA), anti-KAT8 (ab200660, abcam, Cambridge, MA, USA), anti-Acetyl-lysine (PTM-105RM, PTMBIO, China), anti-Flag (F1804, Sigma Aldrich), anti-SIRT1 (8469 S, Cell Signaling Techology, Danvers, MA, USA), anti-SIRT7 (5360 S, Cell Signaling Techology, Danvers, MA, USA), H4K16ac (ab109463, Abcam Cambridge, MA, USA), anti-RNA Pol II (sc-47701, Santa Cruz, CA, USA), anti-E-cadherin (14472 S, Cell Signaling Techology Danvers, MA, USA), anti-N-cadherin (13116 S, Cell Signaling Techology Danvers, MA, USA), anti-Histone H4 (ab177840, Abcam, Danvers, MA, USA), anti-His (PM032, MBL), anti-GST (sc-138, Santa Cruz, CA, USA), anti-GCN5 (sc-365321, Santa Cruz, CA, USA), anti-myc (M047-3, MBL), anti-α-tubulin (BE0031, EASYBIO, Beijing, China) and anti-β-actin (4967, Cell Signaling, Danvers, MA, USA) were used and the blots were developed using an enhanced chemiluminescence kit (Amersham Corp.).

WT and Mincle^−/− GM-CSF BMDMs were differentiated over 8 days, lifted using Accutase (StemCell) and seeded on cover slides coated with iPrOH as a control, 8 nmol/slide AF-2 or TDB, or treated with Nigericin (10 μM) as a control for pyroptosis. After 4 h, the cells were washed with D-PBS (Gibco) and stained with anti-myeloperoxidase (MPO) antibody [2D4] (FITC conjugated, Abcam), anti-histone H4 (acetyl K16) antibody [EPR1004] (Alexa Fluor 647 conjugated, Abcam) and DAPI (BD Pharmagen) for 30 min. After further washing with PBS, ProLong^™ Diamond Antifade Mountant (Invitrogen) was used to mount cover slips onto microscope slides. Confocal images were taken using the inverted microscope IX83 equipped with a confocal head FV3000, employing the blue filter to observe the DAPI dye, red filter for H4 and the green filter to observe the MPO. The BMMs with media only treatment was used to correct for autofluorescence of the cells.

NETs were induced with E. histolytica trophozoites on the Chamber Slide™ System as described above, and the cells were fixed with 4% paraformaldehyde for 10 min. The fixed cells were then permeabilized by adding 0.2% Triton X-100 in PBS for 5 min. Detergent was washed out three times with cold PBS and unspecific protein binding was blocked with a solution of 1% BSA, 0.3 M glycine, 0.1% Tween 20 in PBS for 30 min at 37°C. The samples were incubated with primary anti-NE (Santa Cruz Biotechnology), anti-myeloperoxidase (Abcam), anti-histone H4 (Abcam), or anti-citrulline (Abcam) antibodies diluted 1:100 in 1% BSA, 0.1%Tween 20 in PBS during 1 h at room temperature. The cells were gently washed with cold PBS and incubated with secondary anti-mouse IgG-FITC (Sigma) or anti-rabit IgG-TRITC (Zymax) antibodies diluted 1:50 in the same solution that primary antibody for 1 h at room temperature in darkness. The cells were then washed with PBS and stained with 5 μg/mL DAPI. The coverslips were mounted with Fluoroshield (Sigma) before observation in a fluorescence microscope (Olympus BX51).

Western blot analysis was performed according to a standard protocol. The following antibodies were used to detect each target protein: anti-LDHA (3582 S, Cell Signaling, Danvers, MA, USA), anti-Lamin A (sc-71481), anti-GAPDH (sc-31915), anti-Cytochrome C (sc-13560), anti-β-actin (sc-47778, Santa Cruz, Dallas, TX, USA), anti-Histone H4 (ab10158, Abcam, Cambridge, UK), anti-acetyl-histone H4 (#06-866, Merck, Kenilworth, NJ, USA), anti-mouse (sc-2005), anti-rabbit (sc-2004), and anti-goat IgG-HRP (sc-2922, Santa Cruz, Dallas, TX, USA) antibodies.

Parasite fractions for western blotting were prepared as previously described (Voss et al., 2002 (link)). Samples were loaded onto 4-12% polyacrylamide gels and electrophoresed at 100V for 60 mins. Electrophoretic transfer to nitrocellulose membrane was carried out at 100V for 60 mins. Membranes were blocked in TBST with 5% milk protein and probed with the following antibodies: 1:2000 anti-Ty1 (Invitrogen), then 1:1500 goat anti-mouse IgG-HRP (Dako); 1:1000 anti-HA (Roche), then 1:1500 goat anti-rat IgG-HRP (Biolegend); anti-histone H4 (Abcam), then 1:1000 goat anti-rabbit IgG-HRP (Abcam); or 1:1000 13.3 anti-GAPDH (European Malaria Reagent Repository), then 1:1500 goat anti-mouse IgG-HRP (Dako). Membranes were washed for 3 x 5 mins in TBST after each antibody step. Clarity Western ECL substrate (Bio-Rad) was added for 3 mins and blots were imaged using a FluorChemM chemiluminescent detection camera (ProteinSimple).
Recombinant protein was blotted with anti-His antibody using the same method: 1:2000 mouse anti-tetra-His IgG (Qiagen); 1:1500 goat anti-mouse IgG-HRP (Dako). Coomassie staining of recombinant protein after gel electrophoresis was performed by addition of 0.1% Brilliant blue R-250 for 20 mins (Fisher), then de-staining in 40% methanol 10% glacial acetic acid.

Mice infected by recombinant pMV262/MS, Rv0888NS/MS, D438A/MS, and H481N/MS were anesthetized with diethyl ether, and the trachea was exposed. Subsequently, a closed IV catheter system (BD Intima, Franklin Lakes, NJ, USA) was inserted into the trachea and washed with 500 µl PBS three times. The collected BALF was incubated with 100 U DNase I (Amresco, Solon, OH, USA) at 37°C for 30 min, and the supernatant was analyzed by Western blotting with the following primary antibodies: anti-histone H4 (Abcam, ab17036), anti-histone H3 (citrulline R2 + R8 + R17) (Abcam), and anti-MPO (Abcam, ab139748). Then, incubation with secondary anti-mouse IgG Dylight 680-labeled antibody or anti-rabbit IgG Dylight 800-labeled antibody (KPL, USA) was carried out. Reactivity was detected and recorded on a Li-Cor Odyssey imaging system (Li-Cor Biosciences, USA).