Bolt transfer buffer

To further analyze proteins separated by SDS-PAGE, they were transferred to nitrocellulose membrane using wet blotting and followed by immunodetection. Proteins were transferred in Bolt Transfer Buffer (Life Technologies) for 1 h at room temperature and 10 V. To verify that protein transfer was successful and uniform, membranes were stained with Ponceau solution for 1 min and distained in water until pink bands on a white background were revealed. Membranes were stained with Ponceau followed by washing with Tris Buffered Saline (TBS) for 5 min to remove the Ponceau solution, and then incubated in 5% BSA TBS-Tween (TBS-T) under continuous shaking for 1 h at room temperature to block unspecific binding sites. Incubation with the primary antibody was carried out in 5% BSA TBS-T at the appropriate dilution overnight under continuous shaking at 4°C. To remove unbound primary antibody, membranes were washed three times for 5 min with TBS-T at room temperature. The secondary antibody was diluted in TBS-T and incubated for 1 h before repeating the washing steps. Detection was performed using an Odyssey CLx imaging equipment (LI-COR Biosciences).

Protein samples were resolved via SDS-PAGE using 10% (w/v) or 4–12% (w/v) gradient NuPAGE™ Bis–Tris Protein Gels (Life Technologies Australia Pty Ltd) and stained using Simply Safe Stain (Invitrogen) according to the manufacturer protocol. To conduct western immunoassay proteins in SDS-PAGE gels were transferred to a polyvinylidene difluoride membrane (Life Technologies) using Bolt™ Transfer Buffer (Life Technologies). Membranes were blocked using 5% (w/v) skimmed milk powder in PBS buffer (137 mM NaCl, 10 mM NaH₂PO₄·H₂O, 2.7 mM KCl, pH 7.4) for one hour. The anti-His-tag monoclonal antibody (Sigma-Aldrich) was diluted in PBST buffer (137 mM NaCl, 10 mM NaH₂PO₄, 2.7 mM KCl, with 0.2% Tween-20, pH 7.4) and used to probe the membrane overnight at 4º C in the same blocking solution. The blot was washed four times in 25 mL of PBST buffer for 10 min each then incubated at room temperature for 1 h with rabbit anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (Sigma-Aldrich) in blocking solution. Following three washes in PBST for 15 min each, HRP activity was detected using Immobilon™ Western HRP chemiluminescent substrate with a luminol peroxidase solution according to the manufacturer’s instructions (Millipore Corporation, MA, USA) with image capture using a LAS 3000 Imager (Fujifilm, Japan).

IP elutions were separated on 4–12% gradient NuPage Bolt Bis-Tris gels (Thermofisher) using the XCell SureLock Mini-Cell Electrophoresis System (Thermofisher). Proteins were transferred to a nonfluorescent Immobilon polyvinylidene fluoride membrane (45 μM; Millipore) in Bolt transfer buffer (Thermofisher) using a minitransblot cell (Bio-Rad). After transfer, the membranes were blocked for 1 h at room temperature in a blocking buffer consisting of Odyssey blocking buffer (LI-COR Biosciences) diluted 1:1 with phosphate-buffered saline (PBS). The membranes were incubated overnight at 4 °C in a blocking buffer containing primary antibodies (1:1,000 dilution). After being washed with PBST, the membranes were incubated for 45 min with fluorescently conjugated secondary antibodies diluted in a blocking buffer (1:10,000). Bound antibody was detected by the Odyssey CLx Imaging System (LI-COR Biosciences) using 800 nm and 700 nm channels.

Histones were extracted using the Histone Extraction Kit (Abcam, Cambridge, UK). Protein concentration was quantified by DC Protein Assay (Bio-Rad, Hercules, CA, USA) and 20 µg of protein were loaded on a Bolt 10 % Bis-Tris Plus Gel, 10-Well (Thermo Fisher Scientific) using 4X Bolt LDS sample buffer and 10X Bolt Sample Reducing Agent (both Thermo Fisher Scientific). Electrophoresis war carried out at 200 V for 25–35 min using PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa and Bolt MES SDS Running Buffer (both Thermo Fisher Scientific). Protein was transferred to an Immobilon-FL PVDF membrane (Merck-Millipore) at 50 V for 200 min by using Bolt Transfer Buffer (Thermo Fisher Scientific). After blocking, primary antibodies (Tri-Methyl-Histone H3 (Lys4) from Cell Signaling and Anti-Histone H3 (mono methyl K79) antibody, Anti-Histone H3 (di methyl K79) antibody, Anti-Histone H4 (symmetric di methyl R3) antibody, and Anti-Histone H3 antibody from Abcam), and the secondary antibodies (anti-rabbit IgG, IRDye 680RD, polyclonal and ant-rabbit IgG, IRDye 800CW, polyclonal from Li-Cor Biosciences) were used. Analysis was carried out at the Li-Cor Odyssey CLx using the Image Studio Software (both Li-Cor Biosciences) for documentation. Relative quantification was performed using Histone H3 as the reference protein.