Horseradish peroxidase

Both treated and untreated cells were washed twice with ice‐cold PBS and extracted on ice with cell lysis buffer (KeyGen Biotech Co. Ltd., Nanjing, China). The concentration of the protein was quantified using the BCA Protein Assay Kit (KeyGen Biotech Co. Ltd., Nanjing, China). Thirty micrograms of total protein was electrophoresed through 10% and 12% SDS PAGE and was then transferred to membranes (Millipore, USA). The membranes were blocked in 5% fat‐free milk for two hours then incubated with primary antibodies at 4°C overnight. Secondary antibodies were labelled with Horseradish Peroxidase (Abbkine Scientific, China) and the signals were detected using the ECL Kit (Advansta, UK). Subsequently, the images were analysed using the ImageJ 6.0 software. A glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody was used as the control for whole‐cell lysates.

Pro-inflammatory cytokines interleukin 1β (IL-1β), IL-6, and TNF-ɑ of the hippocampus were evaluated using western blot. We extracted and quantified the total protein of hippocampal tissue. Equal protein amounts were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). Membranes were then blocked in 3% nonfat milk for 1 h at room temperature followed by overnight incubation at 4°C with primary antibodies against IL-1β (ABclonal, Woburn, MA, USA; A1112, 1:1000), IL-6 (ABclonal, Woburn, MA, USA; A0286, 1:1000), TNF-ɑ (ABclonal, Woburn, MA, USA; A11534, 1:1000), and β-actin (Proteintech, Chicago, IL, USA; 66009-1, 1:2000). Next, we incubated the membranes with appropriate secondary antibodies conjugated with horseradish peroxidase (1:10,000; Abbkine, Beijing, China) for 2 h at room temperature. Protein bands were detected using the ChemiDoc MP system (Bio-Rad, Hercules, CA, USA).

Expression levels of glucocorticoid receptor (GR), the DNMTs DNMT1 and DNMT3a, and the DNA demethylase Tet methylcytosine dioxygenase (Tet)-2 in the hippocampus were evaluated by western blotting. Total protein was extracted from hippocampal tissue samples and quantified, and equal amounts were loaded and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, United States) that was blocked in 3% non-fat milk for 1 h at room temperature followed by overnight incubation at 4°C with primary antibodies against GR (Cell Signaling Technology, Danvers, MA, United States; 12041S, 1:1,000), DNMT1 (ab188453, 1:1,000) and DNMT3a (ab188470, 1:1,000) (both from Abcam, Cambridge, MA, United States), Tet-2 (ABclonal, Woburn, MA, United States; A1526, 1:1,000), and β-actin (Proteintech, Chicago, IL, United States; 66,009–1, 1:2,000). The specificity of antibodies was tested using western blotting, which showed that only one band was detected at the expected molecular weight in all antibodies. The membrane was then incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (1:10,000; Abbkine, Beijing, China) for 2 h at room temperature. Protein bands were detected using the ChemiDoc MP system (Bio-Rad, Hercules, CA, United States).