*4.4. Immunoblotting*

Monocultured JEG-3 cells were harvested after washing three times with PBS (Lonza Ltd., Basel, Switzerland). The co-cultured HEC-1A cells were separated from JEG-3 cells by removing the coverslips from the surface of JEG-3 cells containing culture dishes. The coverslips were washed three times with PBS and the cells were collected by trypsinization. The co-cultured JEG-3 cells were collected from the surface of the culture dish using scraper after washing three times with PBS. Pelleted cells of each sample were lysed with 130 µL of M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with Complete mini protease inhibitor cocktail (Roche Ltd., Basel, Switzerland) and PhosSTOP phosphatase inhibitor (Roche Ltd., Basel, Switzerland). The protein contents of the samples were measured with DC Protein Assay Kit (Bio-Rad Inc., Hercules, CA, USA). The same amount of protein (signalling proteins—10 µg, implantation-related proteins—15 µg) from each sample was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% or 12% polyacrylamide gel, and transferred by electroblotting to nitrocellulose membranes (Pall AG, Basel, Switzerland). The membranes were blocked with 5% (*w*/*v*) non-fat dry milk (Bio-Rad Inc., Hercules, CA, USA) solved in TBST (Tris buffer saline, 0.1% Tween-20) for 1 h at room temperature with gentle shaking. After the blocking step, the membranes were incubated with the following polyclonal rabbit antibodies for 1 h at room temperature in case of FineTest primary antibodies and for overnight at 4 ◦C in case of the primary antibodies of Cell Signalling Technology: anti-progesterone receptor A/B IgG (1:1000; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-fractalkine receptor IgG (1:1000; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-activin receptor 1B IgG (1:1000; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-matrix-metalloproteinase 2 IgG (1:2000; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-matrix-metalloproteinase 9 IgG (1:500; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-SRC-1 IgG (1:1000; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-Sox17 IgG (1:1000; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-BMP2 IgG (1:1000; Wuhan Fine Biotech Co., Ltd., Wuhan, China), anti-phospho-AKT IgG (1:2000; Cell Signaling Technology Europe, Leiden, The Netherlands), anti-phospho-JNK (1:1000; Cell Signaling Technology Europe, Leiden, The Netherlands), anti-phospho-ERK1/2 (1:1000; Cell Signaling Technology Europe, Leiden, The Netherlands) and anti-phospho-p38 (1:1000; Cell Signaling Technology Europe, Leiden, The Netherlands). β-actin (1:2000; Merck KGaA., Darmstadt, Germany) was used as the loading control. Goat anti-rabbit IgG, HRP-linked antibody was used as secondary antibody (1:2000; Cell Signaling Technology Europe, Leiden, The Netherlands) for 1 h at room temperature. The detection of the proteins was carried out with WesternBright ECL chemiluminescent substrate (Advansta Inc., San Jose, CA, USA). Optical densities of Western blots were determined using ImageJ software [73] and was expressed as percentage of target protein/β-actin abundance.
