Application

Western Blotting Analysis: 4.2 µg/mL from a representative lot detected 100 nM dexamethasone/100 nM of H2O2 treatment-induced glucocorticoid receptor Ser287 phosphorylation in primary ear fibroblasts from wild-type, but GR S287A-knock-in mice (Courtesy of Elina Sherestha and Michael J. Garabedian, PhD).

Chromatin Immunohistochemistry (ChIP) Analysis: A representative lot detected enhanced Ser287 phosphorylation of GR recruited to the SGK1 and GILZ regulatory regions upon dexamethasone treatment or BDNF/dexamethasone cotreatment of 293TrkB cells stably expressing wild-type rat GR (Lambert, W.M., et al. (2013). Mol. Cell. Biol. 33(18):3700-3714).

Immunofluorescence Analysis: A representative lot detection GR S287 phosphorylation in hypothalamic CRH-producing neurons by fluorescence immunohistochemistry staining of 4% paraformaldehyde-fixed free-floating mouse coronal sections. GR S287 phosphorylation decreased in brain tissue from BDNF+/ and TrkB TrkB+/ mice, increased upon inducible BDNF overexpression and decreased upon Cre-dependent excision in transgenic mice with floxed GR allele (Lambert, W.M., et al. (2013). Mol. Cell. Biol. 33(18):3700-3714).

Western Blotting Analysis: A representative lot detected BDNF-induced GR Ser287 phosphorylation in primary rat cortical neurons, PC12 cells expressing TrkB, and 293TrkB cells stably expressing wild-type rat GR, as well as upregulated GR Ser287 phosphorylation in mouse brain following dexamethasone injection or stress-induced elevation of endogenous glucocorticoids by forced swim (Lambert, W.M., et al. (2013). Mol. Cell. Biol. 33(18):3700-3714).

Detect Glucocorticoid Receptor phosphorylated on Serine287 using this rabbit polyclonal Anti-GR pSer287, Cat. No. ABS1582, validated for use in Western Blotting and Immunofluorescence, Western Blotting, Western Blotting and Chromatin Immunoprecipitation (ChIP).

General description

Glucocorticoid receptor (Uniprot P06536; also known as GR, Nuclear receptor subfamily 3 group C member 1) is encoded by Nr3c1 (also known as Grl, Grc) gene (Gene ID: 24413) in rat species. It has a dual mode of action whereby it acts as a transcription factor that binds to glucocorticoid response elements, both for nuclear and mitochondrial DNA, and also acts a modulator of other transcription factors. GR is composed of several conserved structural elements, including a carboxy-terminal ligand-binding domain (which contains residues critical for receptor dimerization and hormone-dependent gene transactivation), a neighboring hinge region containing nuclear localization signals, a central zinc-finger-containing DNA-binding domain, and an amino-terminal variable region that participates in ligand-independent gene transcription. In the absence of hormone, a significant population of GR is localized to the cytoplasm in an inactive form via its association with regulatory chaperone proteins, such as HSP90, HSP70, and FKBP52. Upon hormone binding, GR is released from the chaperone complex and translocates to the nucleus as a dimer to associate with glucocorticoid response elements (GREs), thereby enhancing or repressing transcription of specific target genes. GR-mediated transcriptional activation is modulated by phosphorylation and upon binding to receptor agonist it becomes hyperphosphorylated. Although basal GR phosphorylation at Ser155 and Ser287 is low, but it is highly inducible by BDNF treatment. It is been reported that BDNF-induced phosphorylation increases GR occupancy and cofactor recruitment at the promoter of a BDNF-enhanced gene.

Immunogen

Synthetic peptide corresponding to rat glucocorticoid receptor target site sequence containing the phosphoryated serine residue.

Other Notes

Concentration: Please refer to lot specific datasheet.

Physical form

Purified rabbit polyclonal antibody in buffer containing TBS, pH 8.0, containing no preservatives.

Quality

Evaluated by Western Blotting in mouse ear fibroblasts.

Western Blotting Analysis: 2.1 µg/mL of this antibody detected 100 nM dexamethasone/100 nM of H2O2 treatment-induced glucocorticoid receptor Ser287 phosphorylation in primary ear fibroblasts from wild-type, but GR S287A-knock-in mice.

Specificity

This polyclonal antibody detected decreased GR S287 phosphorylation in brain tissue from BDNF+/ and TrkB TrkB+/ mice, as well as GR pS287 upregulation upon inducible BDNF overexpression and Cre-dependent GR pS287 downregulation in transgenic mice with floxed GR allele (Lambert, W.M., et al. (2013). Mol. Cell. Biol. 33(18):3700-3714). The target phosphorylation site coresponds to Ser287 of rat GR spliced isoform A and Ser260 of isoform B reported by UniProt (P06536). The equivalent site is also found in human (Ser247 of isoforms Alpha-B & Beta-B, and Ser267 of all the other seven spliced isoforms except GR-P) and mouse (Ser275 of isoform 1-A & 1-B, Ser248 of isoform 2-A & 2-B) GR sequences reported by UniProt (P04150 & P06537).

Target description

~90 kDa observed. 85.66/81.51/85.82/81.67/64.75/60.60/82.85/78.70/82.90 kDa (Human isoform Alpha-A/Beta-A/Alpha-2; Gamma/Beta-2/GR-A alpha/GR-A beta/Alpha-B/Beta-B/hGRDelta313-338), 86.05/86.21/83.27/83.43 kDa (Mouse isoform 1-A/2-A/1-B/2-B), and 87.56/84.83 kDa (Rat isoform A/B) calculated. Uncharacterized bands may be observed in some lysate(s).