To determine whether dexamethasone can induce phosphorylation of GRs in HRMPs, THP-1, and HRECs, we used an anti-phospho-Ser
211 antibody capable of detecting phosphorylation of receptors by Western blot analysis. Dexamethasone-induced phosphorylation of GRs on Ser
211 and subsequent proteasome-mediated receptor degradation have been shown to act as biomarkers for activated GRs in vivo.
26 The day before the experiment, confluent HRMPs, THP-1, or HRECs, growing in 100-mm tissue culture plates (BD Labware, Bedford, MA), were washed once with phosphate-buffered saline (PBS) and stepped to 1% FBS-containing medium with antibiotics-antimycotics. The day of the experiment, the cells were incubated with cell culture–tested dexamethasone (Sigma-Aldrich, St. Louis, MO) at 10, 50, or 100 nM or 1 μM for the indicated times (see
Fig. 3 ). The cells were washed twice with ice-cold PBS and lysed in 400 μL of radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mM EDTA; Upstate Biotech, Lake Placid, NY), supplemented with 0.2% sodium dodecyl sulfate (SDS) and a 1% (vol/vol) cocktail of protease inhibitors, serine-threonine phosphatase inhibitors, and tyrosine phosphatase inhibitors (Sigma-Aldrich). After 30 minutes’ incubation on ice, the lysates were centrifuged at 14,000 rpm for 20 minutes at 4°C. Supernatants were transferred to clean microfuge tubes, and the total protein concentration of each sample was measured with a bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL). Cell lysates (30 μg protein) in SDS sample buffer were separated on 7% Tris-acetate gel by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Phosphorylated GR and total GR levels were detected by using the polyclonal anti-p-GR (Ser
211; Cell Signaling Technology, Beverly, MA) and anti-GR antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Protein bands were visualized by using a chemiluminescence detection kit (Invitrogen, Carlsbad, CA). The intensity of all bands was estimated by densitometric analysis (ImagePro Plus Software; Media Cybernetics, Silver Spring, MD).