Cell Signaling Technology

Product Pathways - Nuclear Receptor Signaling

SignalSilence® Glucocorticoid Receptor siRNA II #7699

Applications Reactivity
Transfection H

Reactivity Key:  H=Human
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® Glucocorticoid Receptor siRNA I #6508 (+), or SignalSilence® Glucocorticoid Receptor siRNA II (+), using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb #3660 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The Glucocorticoid Receptor (D8H2) XP® Rabbit mAb confirms silencing of glucocorticoid receptor expression, while the GAPDH (D16H11) XP® Rabbit mAb is used as a loading control.

Description

SignalSilence® Glucocorticoid receptor siRNA II from Cell Signaling Technology (CST) allows the researcher to specifically inhibit glucocorticoid receptor expression using RNA interference, a method whereby gene expression can be selectively silenced through the delivery of double stranded RNA molecules into the cell. All SignalSilence® siRNA products from CST are rigorously tested in-house and have been shown to reduce target protein expression by western analysis.

Quality Control

Oligonucleotide synthesis is monitored base by base through trityl analysis to ensure appropriate coupling efficiency. The oligo is subsequently purified by affinity-solid phase extraction. The annealed RNA duplex is further analyzed by mass spectrometry to verify the exact composition of the duplex. Each lot is compared to the previous lot by mass spectrometry to ensure maximum lot-to-lot consistency.

Directions for Use

CST recommends transfection with 100 nM SignalSilence® Glucocorticoid Receptor siRNA II 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use.

Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 μl per well.

Background

Glucocorticoid hormones control cellular proliferation, inflammation, and metabolism through their association with the glucocorticoid receptor (GR)/NR3C1, a member of the nuclear hormone receptor superfamily of transcription factors (1). GR is composed of several conserved structural elements, including a carboxy-terminal ligand-binding domain (which also 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. On hormone binding, GR is released from the chaperone complex and translocates to the nucleus as a dimer to associate with specific DNA sequences termed glucocorticoid response elements (GREs), thereby enhancing or repressing transcription of specific target genes (2). It was demonstrated that GR-mediated transcriptional activation is modulated by phosphorylation (3-5). Although GR can be basally phosphorylated in the absence of hormone, it becomes hyperphosphorylated upon binding receptor agonists. It has been suggested that hormone-dependent phosphorylation of GR may determine target promoter specificity, cofactor interaction, strength and duration of receptor signaling, receptor stability, and receptor subcellular localization (3). Indeed Ser211 of human GR is phosphorylated to a greater extent in the presence of hormone, and biochemical fractionation studies following hormone treatment indicate that Ser211-phosphorylated GR is found in the nucleus (3). Thus, Ser211 phosphorylation is a biomarker for activated GR in vivo. An added layer of complexity to GR signaling lies in the ability of multiple isoforms to be generated through both alternative splicing and the use of alternative translation intiation start sites, thus increasing the repertoire of functional signaling homo- and heterodimers (6,7).

  1. Yamamoto, K.R. (1985) Annu. Rev. Genet 19, 209-252.
  2. Necela, B.M. and Cidlowski, J.A. (2003) Trends Pharmacol. Sci. 24, 58-61.
  3. Wang, Z. et al. (2002) J. Biol. Chem. 277, 26573-26580.
  4. Rogatsky, I. et al. (1998) J. Biol. Chem. 273, 14315-14321.
  5. Krstic, M. D. et al. (1997) Mol. Cell. Biol. 17, 3947-3954.
  6. Yudt, M.R. and Cidlowski, J.A. (2001) Mol Endocrinol 15, 1093-103.
  7. Lu, N.Z. and Cidlowski, J.A. (2005) Mol Cell 18, 331-42.

Application References

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Companion Products

Limited Use Label License, RNA interference: This product is licensed under European Patent 1144623 and foreign equivalents from Ribopharma AG, Kulmbach, Germany and is provided only for use in non-commercial research specifically excluding use (a) in drug discovery or drug development, including target identification or target validation, by or on behalf of a commercial entity, (b) for contract research or commercial screening services, (c) for the production or manufacture of siRNA-related products for sale, or (d) for the generation of commercial databases for sale to Third Parties. Information about licenses for these and other commercial uses is available from Ribopharma AG, Fritz-Hornschuch-Str. 9, D-95326 Kulmbach, Germany.

For Research Use Only. Not For Use In Diagnostic Procedures.

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