ABE1424 Sigma-AldrichAnti-Glucocorticoid Receptor Antibody

Hairy and enhancer of split-1 (HES1) is a basic helix-loop-helix transcription factor that is a key regulator of development and organogenesis. However, little is known about the role of HES1 after birth. Glucocorticoids, primary stress hormones that are essential for life, regulate numerous homeostatic processes that permit vertebrates to cope with physiological challenges. The molecular actions of glucocorticoids are mediated by glucocorticoid receptor-dependent regulation of nearly 25% of the genome. Here, we established a genome-wide molecular link between HES1 and glucocorticoid receptors that controls the ability of cells and animals to respond to stress. Glucocorticoid signaling rapidly and robustly silenced HES1 expression. This glucocorticoid-dependent repression of HES1 was necessary for the glucocorticoid receptor to regulate many of its target genes. Mice with conditional knockout of HES1 in the liver exhibited an expanded glucocorticoid receptor signaling profile and aberrant metabolic phenotype. Our results indicate that HES1 acts as a master repressor, the silencing of which is required for proper glucocorticoid signaling.

We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.