4.6. Protein Isolation and Western Blotting

The endometrial tissue explants were homogenized in T-PER Tissue Protein Extraction Reagent (Thermo Fischer Scientific, Waltham, MA, USA) in the presence of peptidase and phosphatase inhibitors (Sigma-Aldrich). The lysates were cleared by double centrifugation at 10,000× g for 5 min. The protein concentrations were measured with the use of Bradford dye-binding procedure with the dilutions of bovine serum albumin (BSA) as standards.

Western blotting analysis was performed as described by Smolinska et al. [14]. Endometrial tissue lysates (40 μg) from control, PGE₂-, and PGF_2α-treated samples were resolved by SDS-PAGE electrophoresis in the 12.5% polyacrylamide gels and transferred onto PVDF membrane (Whatman, USA). Subsequently, membranes were blocked for 1 h in Tris-buffered saline Tween-20 containing 5% skimmed milk powder. After blocking, membranes were incubated for 12 h at 4 °C with rabbit polyclonal antibodies to CMKLR1 (1:1000; ab230442; Abcam, UK), mouse polyclonal antibodies to GPR1 (1:500; ab169331; Abcam, UK), rabbit polyclonal antibodies to CCRL2 (1:600; ab85224; Abcam, UK), and rabbit polyclonal antibodies to actin (1:200; A2066; Sigma-Aldrich, USA). Actin was used as a control to normalize the results of chemerin receptors protein concentration. Subsequently, to identify immunoreactive products, membranes were incubated for 1.5 h at RT with goat anti-rabbit IgG for CMKLR1, CCRL2, and actin (1:5000; sc-2054; Santa Cruz, USA), and goat anti-mouse IgG for GPR1 (1:2500; 115-035-003; Jackson ImmunoResearch Laboratories, Baltimore Pike, PA, USA) conjugated with horseradish peroxidase (HRP). For negative control blots, primary antibodies were substituted by nonspecific fetal calf serum (MP Biomedicals, Santa Ana, CA, USA). Immunocomplexes were visualized with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Kenilworth, NJ, USA) on the G: Box EF Gel Documentation System (Syngene, Cambridge, UK). The same protocol was performed in relation to the adipose tissue used as the positive controls. The results were quantified by densitometric analysis of immunoblots with the use of Image Studio Lite version 5.2 software (LI-COR, Lincoln, NE, USA). Data were expressed as the ratio of chemerin receptors proteins relative to actin protein in arbitrary optical density units.