Granulosa Layer Protein Isolation and Western Blot

The granulosa layers of F5 were used for protein isolation with the BSP003 kit (Sangon Biotech Co., Ltd, Shanghai, China). Protein concentration was measured with the Pierce bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL, USA) and Varioskan Flash instrument (Thermo Fisher Scientific, Rockford, IL, USA). A total of 30 μg protein was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk in 1× Tris-Buffered Saline with Tween (TBST) buffer for 1 h at room temperature, membranes were incubated with rabbit anti-chicken (ERα, ERβ, PR-A, and PR-B) monoclonal antibodies (Abcom, Cambridge, UK, 1:1000) and rabbit anti-chicken β-actin monoclonal antibody (Abcom, Cambridge, UK, 1:1000) overnight at 4°C [27 (link)–29 (link)]. The blots were then washed in 1× TBST buffer and probed with goat-anti-rabbit horseradish peroxidase (HRP)-conjugated IgG secondary antibody (diluted 1:2000 in 1× TBST; Abcom, Cambridge, UK) for 1 h at room temperature. Binding was visualized with enhanced chemiluminescence (ECL) reagent (Beyotime Institute of Biotechnology, Jiangsu, China) using a ChemiDoc XRS instrument (Bio-Rad, Inc., Richmond, CA, USA). Quantity One Software (Bio-Rad, Inc., Richmond, CA, USA) was used for densitometric analysis.