Western-blot immunodetection
Immunodetection was performed by Western-blot after denaturing gel
electrophoresis (Laemmli, 1970) and blotting onto a nitrocellulose
membrane (BioTraceNT 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).