Anti creb

Total proteins were extracted and resolved by SDS-PAGE, transferred to nitrocellulose membranes, blocked by 5% skim milk in 1× TBST buffer, and incubated with the primary antibody at 4 °C overnight. The following primary antibodies were used: anti-GAPDH (Cell Signaling Technology, cat#2118 L, 1:5000), anti-AKT (Cell Signaling Technology, cat#4685 s, 1:2000), anti-phospho-AKT (Cell Signaling Technology, cat#4060 s, 1:2000) anti-CREB (Proteintech, cat#12208-1AP, 1:1000), anti-phospho-CREB (Cell Signaling Technology, cat#9198 S, 1:1000), anti-estrogen receptor alpha (Abcam, cat#ab32063, 1:1000). HRP-conjugated secondary anti-rabbit IgG (Santa Cruz, cat#sc-2357, 1:10,000) was next incubated for 1 h at room temperature. Signals were visualized with ECL Reagent (ShareBio, cat#SBWB012) using GelDoc XR (Bio-Rad).

Zebrafish brains were lysed in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 150 mM NaCl, 1% NP-40, 2 mM ethylenediaminetetraacetic acid, 10 mg/ml leupeptin, 10 mM sodium fluoride, 2 mM sodium orthovanadate, 0.25% sodium deoxycholate, and 2 mM phenylmethylsulfonyl fluoride. Five fish from each treatment were used for the analysis. Lysates were separated using a 10% acrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 1% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20 (PBST) and incubated with anti-phospho ERK and anti-ERK (polyclonal; 1/1,000 dilution; Cell Signaling Technology, MA, United States), anti-β-actin (clone 2D4H5; 1/1,000 diluted; Proteintech, IL, United States), and anti-phospho CREB and anti-CREB (monoclonal, 10E9, D-12, respectively. 1/500 dilution; Santa Cruz Biotechnology, TX, United States). After incubation with a horseradish peroxidase-conjugated secondary antibody, target protein bands were detected using EzWestLumi plus chemiluminescence reagent (ATTO, Tokyo, Japan) using ChemiDoc Touch Plus (Bio-Rad, CA, United States). Densitometric analysis was performed using Image Lab Touch software (Bio-Rad).

BPS (99% purity) was purchased from Sigma-Aldrich (Shanghai, China). Sesame oil (Jinlongyu, Shenzhen, China) was stored in glass bottles without bisphenol compounds. Phenylmethylsulfonyl fluoride (PMSF), 4% paraformaldehyde, 2.5% glutaraldehyde, BCA kit and anti-BDNF (Cat No: GB11559) antibody were purchased from Servicebio Technology (Wuhan, China). Anti-TrkB (Cat No: 13129-1-AP), anti-β-actin (Cat No: 20536-1-AP) and anti-CREB (Cat No: 12208-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA). Anti–phosphorylated (p)-CREB (Cat No: ab32096), anti-DNMT3a (Cat No: ab188470) and anti-DNMT3b (Cat No: ab2851) antibodies were purchased from Abcam (Shanghai, China). Anti-DNMT1 (Cat No: 5032) antibody was purchased from CST (Wuhan, China). The tissue genomic DNA extraction kit (Cat No: DP304) was purchased from Tiangen Biotech (Beijing, China). The other chemicals used in this study are commercially available at the required grade.