Histone h3 antibody

CEK cells were infected with identical doses (200 μL of PBS containing 10⁸ copies) of rYN or rYN-Δ5a. At 12, 24, 36, or 48 hpi, the cells were washed with cold PBS and harvested in 80 μL of 2× lysis buffer. After being incubated on ice for 30 min, the cells were sonicated for 1 min and then boiled. Equal protein amounts were separated by SDS-PAGE, and proteins were detected via Western blotting using specific antibodies against IBV N (Hytest, Finland), β-actin (Merck Millipore, Burlington, MA, USA), α-tubulin antibody (Abcam, Cambridge, UK), and histone H3 antibody (Abcam). Images were taken with the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Protein signal intensities were normalized and quantified using ImageJ software (NIH, Bethesda, MD, USA).

Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium, trypsin (with EDTA), antibiotics (50 U/mL penicillin; 50 μg/mL streptomycin), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from HyClone Laboratories Inc. (Logan, UT, USA). PA, β-hydroxybutyrate, N-acetylcysteine (NAC, an antioxidant), and pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). p-IκBα, IκBα, NF-κB p65, and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and histone H3 antibody was purchased from Abcam (Cambridge, MA, USA). Cell culture flasks were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and six-well plates, 96-well plates, and filters were purchased from Nalge Nunc (Rochester, NY, USA).

CEK cells were infected with identical doses (200 μL of PBS containing 10⁸ copies) of rYN or rYN-Δ5a. At 12, 24, 36, or 48 hpi, the cells were washed with cold PBS and harvested in 80 μL of 2× lysis buffer. After being incubated on ice for 30 min, the cells were sonicated for 1 min and then boiled. Equal protein amounts were separated by SDS-PAGE, and proteins were detected via Western blotting using specific antibodies against IBV N (Hytest, Finland), β-actin (Merck Millipore, Burlington, MA, USA), α-tubulin antibody (Abcam, Cambridge, UK), and histone H3 antibody (Abcam). Images were taken with the ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA). Protein signal intensities were normalized and quantified using ImageJ software (NIH, Bethesda, MD, USA).

ChIP experiments were performed following the procedures described by Wei et al. in 2018 [30 (link)]; the detailed protocol is shown in the supplementary material (File S1). Briefly, at 45 days of age, the thalli were collected and treated with 1.00% formaldehyde for DNA/histone cross-linking for 30 min. After quenching the cross-linking reaction with 2M glycine, we froze the cross-linked thalli samples with liquid nitrogen and ground them. Chromatin was isolated by sucrose density gradient centrifugation. Histone H3 antibody (ab176842, Abcam, Cambridge, UK) and Magna ChIP™ Protein A+G Magnetic Beads (16–663, Sigma-Aldrich, St. Louis, MO, USA) were used for immunoprecipitation in this study. ChIP DNA was purified using the Universal DNA Purification Kit (DP214-02, Tiangen). The purity and integrity of ChIP-extracted DNA were analyzed using agarose gel electrophoresis. The concentrations of ChIP DNA samples were quantified by Qubit. Paired-end sequencing libraries were prepared, as instructed by the Illumina standard protocol, and then loaded on the Illumina HiSeq 2000 platform for sequencing. For one of the dehydrated samples, we generated approximately double the amount of ChIP data (about 13.7 Gb) in order to investigate the effect of data size on genome coverage.

THP1 cells were treated as indicated with NG, HG, PA, and HP and centrifuged at 100g for 10 minutes at 4°C. Cell pellets were washed with cold PBS and lysed in Nuclear Isolation Buffer (NEB) containing 0.25M sucrose, 8 mM Tris HCl (pH 7.4), 5 mM MgCl₂, 0.8% Triton X-100, and 1× Complete Protease inhibitor (Roche) on a rotator for 20 minutes at 4°C. Cell lysates were centrifuged at 2500g for 20 minutes at 4°C; nuclear pellets were lysed in Laemmli sample buffer (without dye and β-mercaptoethanol) and were briefly sonicated (15 seconds at 4°C; Diagenode) to reduce viscosity. Protein concentrations were estimated using Protein Assay kit (Bio-Rad), and equal amounts of nuclear proteins were subjected to Western blotting with G9a antibody (1:1000, catalog 688851S, Cell Signaling Technology) and internal control Histone H3 antibody (1:3000, catalog ab1791, Abcam) (22 (link)). Intensities of protein bands detected by enhanced chemiluminescence were quantified using a GS-900 calibrated densitometer and Image Lab software (Bio-Rad). Results were expressed as the ratio of G9a/H3.

Total proteins were extracted from the top sixth leaves [51 (link)], the cytoplasmic fraction, and the nuclear fraction [52 (link)] of 50-day-old plants grown in the chamber. Western blots were hybridized with the specific antibody against YFP (Novagen) or the specific NtTTG2 antibody, which was produced by immunizing a New Zealand white rabbit in our previous study [47 (link)]. PEPC and histone H3 that were used as cytoplasmic and nuclear markers [4 (link), 41 (link)] were hybridized with a specific PEPC antibody (Rockland) and histone H3 antibody (Abcam), respectively. Hybridized blots were probed by using a horseradish peroxidase-conjugated secondary antibody (Beyotime) according to the manufacturer’s recommendations.