Dnmt3b

Western blot was performed^{51 (link)} to measure the protein expression levels of DNMTs in the PFC. Primary antibodies were rabbit DNMT1 (1:1000, #5032; Cell Signaling Technology), DNMT3a (1:1000, #2160; Cell Signaling Technology), DNMT3b (1:2000, NB300-516; Novusbio, Littleton, CO), brain-derived neurotrophic factor (1:500, sc-546; Santa Cruz Biotechnology), and mouse β-actin (1:10,000, A1978; Sigma-Aldrich). The membranes were then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2000; Pierce, Rockford, IL) and anti-mouse antibody (1:30,000 for β-actin, Pierce). Signals were visualized using a chemiluminescence kit (Super Signal West Pico; Pierce) and intensity was measured by densitometry.

HT22 cells were cultured to 70% confluence in culture dishes with 5% CO₂ and 95% air at 37°C. BpV (0.3 or 3 μM) was added for 12 or 24 hours. After harvesting, cells were washed in phosphate-buffered saline before lysing with radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China). Protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Roche Diagnostics, Indianapolis, IN, USA). Membranes were blocked with 5% nonfat milk before incubation with rabbit polyclonal anti-DNMT1, -DNMT3A, -DNMT3B, or -P21 (a cyclin-dependent kinase inhibitor) (Novus Biologicals, Littleton, CO, USA), and a mouse monocloncal anti-actin antibody (Sigma) (Liu et al., 2017). Primary antibodies were used at a dilution of 1:1000. Blots were visualized with an enhanced chemiluminescence detection system (Beyotime Biotechnology). Optical densities of bands were quantified using ImageJ 1.8.0 software (Scion Corporation, Torrance, CA, USA). Western blot data were normalized to β-actin. Cells were harvested and lysed. Protein expression levels of DNMT1, DNMT3A, DNMT3B, and p21 were determined by immunoblotting analysis as previously described by He et al. (2013).

The NCI–H1299 as well as Calu-3 cells, were crosslinked at 37 °C in 1 % formaldehyde for a period of 10 min. Consequently, for a period of 5 min, they were quenched with glycine. This aligns with the directions provided by the EZ-Magna ChIP® G ChIP kit (17–409, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Correspondingly, the cells were lysed on ice as well as subjected to ultrasonication to produce a chromatin truncation of 200–1000 bp. DNMT1 (1:50, 5032, Cell Signaling Technology, Beverly, MA, USA), DNMT3A (1:50, 3598, Cell Signaling Technology), as well as DNMT3B (1:20, NB300-516, Novus Biologicals) antibodies were then incorporated to the lysates and left overnight at 4 °C. In order to determine the abundance of FZD5 promoter fragments, the protein-chromatin complexes were gathered via magnetic beads and de-crosslinked. Lastly, the DNA was eluted as well as purified for qPCR analysis.