Anti gr antibody

A549 cells were harvested and homogenized in lysis buffer (20 mm HEPES-KOH, pH 7.4, 0.1 m potassium acetate, 2 mm MgCl₂, 0.1% Tween 20, 1 μm ZnCl₂, 1 μm CaCl₂, 0.5% Triton X-100, 250 mm NaCl, 250 units/sample DNase (Novagen), 1/100 (v/v) protease and phosphatase inhibitor cocktails (Roche)) and centrifuged at 8000 × g for 10 min at 4 °C. Lysates were cleared by centrifugation at 13,000 × g at 4 °C and incubated with anti-GR antibody (BD Bioscience) for 1 h at 4 °C. The immunoprecipitation was then performed by incubating cell lysates for 30 min at 4 °C with 10 μl/sample of Dynabeads M-280 sheep anti-mouse IgG (Invitrogen). After 3× more washes in lysis buffer, proteins were eluted in NuPAGE LDS sample buffer (4×) (Invitrogen) containing 100 mm DTT (Invitrogen) and incubated for 10 min at 70 °C. Immunoprecipitated proteins were either frozen at −20 °C or used for immunoblot analysis (as described above) using antibodies against GR, phosphorylated GR, and Merm1.

NHEKs were treated with cortisol with or without mifepristone, eplerenone, or 7,3’,4’-THIF, stained with anti-GR antibody (BD Bioscience, San Jose, CA, USA) or anti-MR antibody (Abcam, Cambridge, MA, USA), and analyzed under a confocal microscope (LSM 700; Carl Zeiss, Jena, Germany). The images obtained were quantified for mean fluorescence intensity (MFI) using ImageJ software (https://imagej.nih.gov/ij). For immunohistochemical analysis, replicate sections from an RHE were stained with the following antibodies: anti-keratin 10 (Biolegend, San Diego, CA), anti-keratin 1 (Biolegend), and anti-filaggrin (Abcam). The detailed protocols for the immunological analyses are described in the Supplementary Information.