Western blot.
We used a protocol previously used in our laboratory (Cuitavi et al., 2021). 1-mm acrylamide gels at 10% with 15 wells were used for the electrophoresis. We mixed 20 µg of total protein, loading buffer (350 mM Tris pH 6.8, 30% glycerol, 30% mercaptoethanol, 100g/L sodium dodecyl sulphate, and 200 mg/L bromophenol blue) and water to obtain the same total protein and volume per sample, and they were heated at 70ºC for 20 minutes. We used the Bio-Rad Mini-PROTEAN buffer system (6 g/L Trizma base, 2.88 g/L glycine, and 20 g/L sodium dodecyl sulphate) to perform the electrophoresis at 120 V for 80 to 90 minutes. Once, the proteins were separated by sodium dodecyl sulphatepolyacrilamide gel electrophoresis (SDS-PAGE), we transferred it to nitrocellulose membranes (Bio-Rad) with the appropriate buffer (3 g/L Trizma base, 1.44 g/L glycine, and 20% methanol) and a semidry system (Bio-Rad Trans-Blot TurboTM) for 25 minutes at 25 V buffer.
Then, membranes were blocked using 5% non-fat dried milk in TBS-Tween 20 (TBS-T) 0.1% (20 mM Tris and 500 mM NaCl pH 7.5) for 1 hour, and just before they were incubated with the corresponding primary antibody (rabbit anti-KOR 1:2000, REF #44-302G, Thermo Fisher; goat anti-CRFR1 1:1800, REF #PA5-18801, Thermo Fisher; rabbit anti-GAPDH 1:2000, Thermo Fisher) overnight at 4º C. After that, membranes were washed 3 times with TBS-T 0.1% and were incubated for an hour at room temperature with the secondary antibody (Goat anti-Rabbit IgG (H+L) HRP conjugated at 1:2000 for GAPDH and KOR (REF #31460, Thermo Fisher; Donkey anti-Goat IgG (H+L) HRP conjugated 1:2000 for CRF1R, REF #A15999, Thermo Fisher). Finally, we developed the membranes with chemiluminescence using CheLuminate-Horseradish peroxidase (HRP) PicoDetect (Panreac, Barcelona, Spain), and images were captured with the ChemiDoc XRS1 System (Bio-Rad) and quantified using ImageJ software. The grey intensity of the bands was normalised dividing the values of the protein of interest by the GAPDH values. The relative protein levels were expressed as mean ± SEM.