Immobilon chemiluminescence reagent

Western blot to detect VEGF expression was conducted as described elsewhere with modifications [30] (link). To separate proteins with SDS-PAGE, a resolving gel with 12% acrylamide was used. After blotting the gel, the PVDF-membrane (Carl Roth GmbH) was blocked with 4% skim milk in Tris buffered saline with 0.1% Tween for 1 hour at room temperature. The blot was treated with the first antibodies, against beta-actin (Cell Signaling Technologies) or VEGF (A-20) (Santa Cruz Biotechnology), overnight at 4°C in 2% skim milk in Tris buffered saline with 0.1% Tween. The VEGF antibody used detects intracellular VEGF containing a signal peptide which initiates export across the endoplasmic reticulum; this signal peptide is cleaved before secretion. After washing, the blot was incubated with anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technologies) in 2% skim milk in Tris buffered saline with 0.1% Tween. Following the final washing, the blot was incubated with Immobilon chemiluminescence reagent (Millipore, Schwalbach, Germany) and the signal was detected with MF-ChemiBis 1.6 (Biostep, Jahnsdorf, Germany). The density of the bands was evaluated using Total lab software (Biostep) and the signal was normalized for β-actin.

Decidual tissue was homogenized on ice in tissue protein extraction reagent (78510, Thermo, Waltham, MA, USA) with proteinase inhibitors (HY-K0010, MCE, Monmouth Junction, NJ, USA) and phosphatase inhibitors (4906837001, Roche, Basel, Switzerland). The protein lysate was placed on ice for 20 min and centrifuged at 14,000 rpm for 20 min at 4 °C. The extracted supernatants were then harvested for subsequent testing. The protein concentration was detected using a BCA protein assay kit (23227, Thermo, Waltham, MA, USA). Proteins were separated using sodium dodecyl sulphate Tris-glycine gels (Bio-Rad), transferred onto PVDF membranes, blocked in 5% non-fat milk, and hybridized overnight at 4 °C with primary antibodies. The primary antibodies included ER-α (1:1000, ab32063, Abcam, Cambridge, UK) and Actin (1:1000, 4970S, CST, Danvers, MA, USA). The secondary antibody that we used was HRP-labeled goat anti-rabbit IgG (1:3000, 7074P2, CST, Danvers, MA, USA). The membranes were washed with Tris-buffered saline containing 0.01% Tween-20. Protein bands were visualized using Immobilon Chemiluminescence Reagent (WBKLS0500, Millipore, Billerica, MA, USA) and analyzed using a ChemiDox Gel imaging system (Bio-Rad, Hercules, CA, USA).