Western-blot immunodetection
Immunodetection was performed by Western-blot after denaturing gel electrophoresis (Laemmli, 1970) and blotting onto a nitrocellulose membrane (BioTraceNT Pall Corporation, East Hills, NY, USA), using a semi-dry procedure (Trans Blot SD Semi-Dry Electrophoretic Transfer Cell; BioRad, Hercules, CA, USA). Membranes were incubated overnight at 4ºC with the primary antibodies (α-GR (AS06 181), dil. 1/5000; α−γECS (AS06 186), dil. 1/2500 (Agrisera, Vännäs, Sweden). After incubation with the anti-rabbit secondary antibody linked to horse radish peroxidase (HRP), proteins were detected using the LumiSensor Chemiluminescent HRP Substrate Kit (GenScript, Piscataway, NJ, USA), and images were taken with a ChemiDoc™ XRS+ System (BioRad).