Erα sc 8002

Lysates from cells and tumor tissues were prepared and protein determined using the BCA assay (Beyotime Biotechnology). Protein (30 µg) from each sample was resolved on SDS-PAGE, and transferred to PVDF membrane. Membranes were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 and incubated with primary antibodies overnight at 4°C. Membranes were washed thrice, 5 min each time, and then incubated with either horseradish peroxidase (HRP)-conjugated goat anti-mouse (ab6789, Abcam, Cambridge, UK) or HRP-conjugated goat anti-rabbit (ab6721, Abcam) secondary antibodies for 2 h at room temperature. Immunoreactive bands were visualized using Pierce ECL plus Western blotting substrate (Thermo Fisher Scienti c, Waltham, MA, USA). The primary antibodies used were: ERα (sc-8002, Santa Cruz Biotechnology, Dallas, TX, USA), HSP90 (ab203126, Abcam), β-actin (4970S, Cell Signaling Technology, Boston, MA, USA), Bax (2774S, Cell Signaling Technology), Bcl-2 (15071S, Cell Signaling Technology), ubiquitin (3936S, Cell Signaling Technology), CYP1A1 (ab124295, Abcam) and AhR (ab190797, Abcam). The protein bands were analyzed using ImageQuant 5.2 software. β-actin was used as loading control.

After treating cells with test compounds for 24 or 48 h, cells were lysed in lysis buffer. Proteins were separated in 4-20% Mini-Protean TGX System Precast Protein Gels (Bio-Rad, Hercules, CA, USA) and transferred to Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad) using a Trans-Blot Turbo Transfer System apparatus (Bio-Rad). The blots were blocked for 1 h in 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 and then incubated overnight at 4 °C with antibodies specific for caspase-3 and cleaved caspase-3 (#9662, Cell Signaling Technology), PARP (#9542, Cell Signaling Technology), ERα (sc-8002, Santa Cruz Biotechnology), ERβ (sc-8974, Santa Cruz Biotechnology), and GPR30 (ab3974, Abcam). The membranes were then washed three times in TBST (Tris-buffered saline, 0.1% Tween 20) and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated antirabbit (#7074) secondary antibodies (Cell Signaling Technology). β-Actin (Cat. No. A5316, Sigma-Aldrich) was used as a loading control. Immunopositive bands were visualized using WesternBright Quantum HRP substrate (Cat. No. K-12043 D20, Advansta Inc., Menlo Park, CA, USA). Quantification of protein bands was performed by densitometry using VisionWorks LS Acquisition and Analysis software (UVP, LLC, Upland, CA, USA).