Anti histone h3

Chp1CD wild-type and mutant nucleosome-binding assays were performed as described earlier for the Chp1CD—H3KC9me3 MLA Nucleosome complex formation in 20 mM HEPES pH 7.5, 100 mM KCl, 0.5 mM DTT, 40 mM imidazole. Resins and INPUTs were run on SDS-polyacrylamide gel electrophoresis 15% acrylamide gels. All samples were then analyzed by immunoblot with anti-H3 histone (AbCam, Cambridge, UK, 1:1000), or anti-H3K9me3 Antibody (AbCam, 1:1000), anti-goat IgG-HRP (BioRad, 1:3000) anti-rabbit IgG-HRP (BioRad, Munich, Germany, 1:3000).

The binding assay of all Clr4 constructs and the three types of nucleosomes were conducted as the following: 0.5 μg of protein were bound to 15 μl of anti-FLAG M2 affinity gel resin (Sigma-Aldrich) in binding buffer (50 mM phosphate buffer pH 6.8, 150 mM NaCl, 1 mM DTT) for 20 min at 4°C. The resin was washed once with the binding buffer and 2 mg of nucleosomes were then added to reach a final volume of 40 μl. After 1 h incubation on ice, the resin was centrifuged and the flow-through was collected. The flow-through, resins and inputs were analyzed on SDS-PAGE 15% acrylamide gels then silver staining (0.1% AgNO₃). All samples were further analyzed with western blot using anti-H3 histone (AbCam, 1:1000), anti-H3K9me3 Antibody (AbCam, 1:1000), anti-goat IgG-HRP (BioRad, 1:3000) and anti-rabbit IgG-HRP (BioRad, 1:3000).
The Clr4-H3KC9me3 nucleosome complexes (Clr4 FL, Clr4 CD and Clr4 1–191 constructs) were prepared as mentioned above using 5 μg protein and 10 μg nucleosomes and eluted after incubation with FLAG peptide (Sigma-Aldrich).

Strains were grown in 100 mL ANM liquid medium with supplements and 10 mM ammonium tartrate at 37 °C for 16 h. For nitrogen starvation, mycelia were washed with and transferred to pre-warmed nitrogen-free ANM media for the indicated amount of time. Xylose was added to the media at a final concentration of 1% to induce PnmB and PrtA overexpression in the respective overexpression strains. Mycelia were harvested on Miracloth, pressed dried, snap-frozen in liquid nitrogen, and kept at −80 °C until protein extraction. Total proteins were extracted as previously described^{19 (link)}. In all, 50 μg of total proteins were separated in 10% SDS-PAGE gel and transferred to PVDF membrane for immuno-detection. PnmB^HA, NmrA^FLAG, and histone H3 were detected using HA-probe (Santa Cruz sc-7392), anti-FLAG® M2 (Sigma F1804), and anti-Histone H3 (Abcam ab1791) antibodies, respectively, at the concentration of 0.1 µg/mL. Goat anti-mouse (Millipore AP124P) or goat anti-rabbit horseradish peroxidase (HRP)-conjugated (Millipore AP132P) antibodies were used as the secondary antibody at the concentration of 0.1 µg/mL for Chemiluminescence detection using Clarity™ Western ECL Substrate (Bio-rad 1705060) kit.

HEK293T and SHSY5Y cells were grown and maintained in Dulbecco’s modified Eagle’s medium (Gibco) at a 37 °C incubator supplied with 5% CO₂. Dulbecco’s modified Eagle’s medium was supplemented with 10% fetal bovine serum (Gibco), 1% antibiotic–antimycotic (Gibco), and 1% essential amino acids (Gibco). Untransfected and FLAG-TSPYL5–transfected HEK293T cell pellets were resuspended in hypotonic buffer (10 mM Hepes [pH 7.4], 10 mM NaCl, 6 mM MgCl₂, 2 mM DTT, 0.05% NP-40, 2 mM PMSF, and 1× protease inhibitor cocktail), incubated on ice for 1 h, and centrifuged at 3000 rpm for 5 min. The supernatant fraction was collected as a cytosolic fraction. The pellet fraction was treated with DNase I in nuclear extraction buffer (20 mM Hepes [pH 7.4], 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 20% glycerol, and 0.5% NP-40) and incubated at 37 °C for 30 min followed by centrifugation at 13,000 rpm for 10 min at 4 °C. The supernatant fraction thereafter collected was marked as a nuclear fraction, and the pellet was discarded. Fractionation was analyzed by Western blotting using anti-TSPYL5, antitubulin (Bio-Rad; catalog no.: MCA78G) as a cytosolic marker, and anti–histone H3 (Abcam; catalog no.: ab10799) as a nuclear marker.

All general reagents and chemicals were purchased from Sigma–Aldrich, unless otherwise specified. Supplements and media for cell culture were from Invitrogen. Antibodies were purchased from Cell Signaling Technology (anti-Sirt6, catalog no.: 12486; anti-p27, catalog no.: 3688; anti–histone H3, catalog no.: 4620; and antiubiquitin, catalog no.: 3936), Abcam (anti-TNFα, catalog no.: ab183218 and anti-Sirt6, catalog no.: ab191385), or Sigma–Aldrich (anti–beta actin, catalog no.: A5441 and anti–alpha tubulin, catalog no.: T6074). Antibodies for flow cytometry were purchased from Thermo Fisher Scientific (LIVE/DEAD Fixable Far Red Dead Cell Stain Kit; catalog no.: L10120), BioLegend (CD19-APCCy7, catalog no.: 115530; T-cell receptor beta (TCRβ)-APCCy7, catalog no.: 109220; Ly6G-APCCy7, catalog no.: 127624; CD11b-BV510, catalog no.: 101245; and ZOMBIE-AQUA, catalog no.: 423102), or Millipore (F4/80-PE, catalog no.: MABF1530).