Anti erβ

The human LUAD cell lines (A549, H1975, and H-1299) and the breast carcinoma cell lines (MCF-7 and MD-MBA-231) used in this study were purchased from the Cell Bank of the Chinese Academy of Sciences. Cell line authenticity was verified using STR analysis and mycoplasma was tested by the supplier prior to their shipment. The cells were maintained in DMEM medium (Gibco, Cat# 11995065) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Biological Industries) at 37°C with 5% CO₂. Before E₂ treatment, the medium was changed to complete phenol-red free DMEM (Gibco, Cat# 21063029) supplemented with 5% charcoal stripped FBS (Hyclone, Cat# SH30068.03). 17β-Estradiol (E₂) was purchased from Sigma-Aldrich (Cat# E2257). E₂ was dissolved and stocked in ethanol at the concentration of 1 mM before addition into the culture medium. When being used for the treatment of cells, the final volume ratio of E₂-ethanol solution to the medium was no more than 1:10,000 (V:V). The primary antibodies used in this study were purchased as follows: anti-PELP1, from Bethyl (Cat# IHC-00013); anti-ERα, from Santa Cruz (Cat# sc-543); anti-ERβ, from Abcam (Cat# ab288); anti-PCNA, from Boster (Cat# BM0104); anti-MMP-9, from Abcam (Cat# ab76003); anti-β-Actin, from Santa Cruz (Cat# sc-69879).

Western blotting was performed as previously described (52 , 54 (link)). Briefly, the cells and tissues were harvested and lysed using RIPA lysis buffer. The protein sample were separated using 4 to 20% SDS-PAGE and transferred to polyvinylidene difluoride membrane. Following blocking with 5% nonfat milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies at 4 °C overnight: anti-ERα (1:1000) (Cat. No. ab32063, Abcam), anti-ERβ (1:1000) (Cat. No. PA1-311, ThermoFisher) antibody, and anti-GAPDH (1:10,000) (Cat. No. 60004-1-Ig, Proteintech), anti-β-catenin (1:1000) (Cat. No. 8480, Cell Signaling Technology), anti-Cyclin D1 (1:1000) (Cat. No. 2978, Cell Signaling Technology), anti-PCNA (1:1000) (Cat. No. ab29, Abcam). Then, the membranes were washed with Tris-buffered saline with 0.1% Tween 20 detergent three times and incubated with corresponding secondary antibodies for 2 h at room temperature. The signals were visualized using enhanced chemiluminescence (Millipore) in an ImageQuant LAS 400 digital biomolecular imaging system. Each experiment was repeated three times. The gray value of the bands was measured by ImageJ software for statistics.

The protein of brain tissues from human and mouse were extracted, and protein concentration was measured using a BCA protein assay. Extracted protein was mixed with 5× loading buffer, electrophoresed in 10% sodium dodecyl sulfate polyacrylamide gel, and transferred onto a polyvinylidene difluoride membrane. The membranes were immersed in 5% skim milk for 2 h at room temperature. After washing with TBST, the membranes were incubated overnight at 4 °C with the primary antibodies. The primary antibodies used in this study were as follows: anti-GAPDH (1:1000, Abcam), anti-ERβ (1:500; from the Jan-Ake Gustafsson's laboratory, Karolinska Institute, Novum, Sweden), anti-VGAT (1:1000, Abcam), anti-VGluT1 (1:1000, Abcam), and anti-Glutamine Synthetase (1:1000, Abcam). After washing, the membranes were incubated with the anti-rabbit or anti-mouse secondary antibodies for 2 h at room temperature. The specific protein bands were visualized in membranes by the chemiluminescence method (Amersham, Piscataway, NJ), and the optical density of protein bands were analyzed in Bio-Rad Image-Lab 6.0 software.