Western blot analysis

Protein extracts were obtained by lysing cells in RIPA buffer (Merck, 20-188, completed with protease inhibitor cocktail, Sigma, 04693132001). Proteins were separated by SDS-PAGE and electro-transferred onto PVDF membranes. Western Blots were placed for blocking in Tris-buffered saline (TBST; 50 mM Tris-Cl, pH 7.6; 150 mM NaCl, 0.05% Tween 20) and 5% non-fat milk for 1 h at room temperature and subsequently incubated with primary antibody TBST overnight at 4°C. The following primary antibodies were used: FKBP5/FKBP51 (1:1,000, Bethyl, A301-430A), MR (1:800, Santa Cruz, N-17), GR (1:800, Cell Signal, #12041), FLAG (1:5,000, Sigma, F3165), HA (1:8,000, 11867423001) and Actin (1:5,000, Santa Cruz, I-19). Subsequently, the blots were washed with TBST and probed with the respective horseradish-peroxidase or fluorophore-conjugated secondary antibody for 2 h at room temperature.

The immuno-reactive bands were detected either by using ECL detection reagent (Millipore, WBKL0500) or directly by excitation of the respective fluorophore. Recording of the band intensities was performed with the ChemiDoc MP from Bio-Rad. Protein data were normalized to Actin, which was detected on the same blot in the same lane (multiplexing).