Cell Signaling Technology

Product Pathways - Nuclear Receptor Signaling

Glucocorticoid Receptor (D8H2) XP® Rabbit mAb #3660

sc-1002   sc-1003   sc-8992  

No. Size Price
3660S 100 µl ( 10 western blots ) ¥3,580.00 现货查询 购买询价
3660T 20 µl ( 2 western blots ) ¥1,400.00 现货查询 购买询价
3660 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human,Mouse,Rat,Monkey, Endogenous 80, 91, 94 Rabbit IgG
IP 1:100
F 1:200
IF-IC 1:100
ChIP 1:100

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, F=Flow Cytometry, IF-IC=Immunofluorescence (Immunocytochemistry), ChIP=Chromatin IP,

Specificity / Sensitivity

Glucocorticoid Receptor (D8H2) XP® Rabbit mAb recognizes endogenous levels of total glucocorticoid receptor protein. Based upon sequence alignment, this antibody is predicted to cross-react with all known alternative translation start site generated isoforms of glucocorticoid receptor-α and glucocorticoid receptor-β. This antibody does not cross-react with mineralocorticoid receptor.Glucocorticoid Receptor (D8H2) XP® Rabbit mAb识别内源性水平的总糖皮质激素受体蛋白。基于序列比对,预测该抗体与所有已知的选择性翻译起始位点产生的糖皮质激素受体-α亚型和-β亚型可以发生交叉反应。该抗体不与盐皮质激素受体发生交叉反应。

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu378 of human glucocorticoid receptor protein.该单克隆抗体通过用合成肽免疫动物制备,该合成肽是人糖皮质激素受体蛋白亮氨酸(378位)附近的残基。

IF-IC

IF-IC

Confocal immunofluorescent analysis of HeLa cells, grown in phenol red-free media containing 5% charcoal-stripped FBS for 2 days and either untreated (upper) or dexamethasone-treated (100 nM, 2hr; lower), using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).激光共聚焦免疫荧光法检测HeLa细胞,细胞在不含酚红,含5%活性炭/葡聚糖处理FBS的培养基中生长2天,之后,细胞不处理(上图)或用地塞米松(100 nM, 2hr; 下图)处理,使用的抗体为Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (绿色). 肌动蛋白丝用DY-554鬼笔环肽标记(红色)。

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb.Western blot方法检测多个细胞系提取物,使用的抗体为Glucocorticoid Receptor (D8H2) XP® Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, either mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human glucocorticoid receptor-α (hGRα-Myc/DDK, +) or Myc/DDK-tagged full-length human mineralocorticoid receptor (hMR-Myc/DDK, +), using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (upper) and DYKDDDDK Tag Antibody (Binds to same epitope as Sigma's Anti-FLAG® M2 Antibody) #2368 (lower).Western blot方法检测293T细胞提取物,细胞不转染或用一个表达Myc/DDK-标记人全长糖皮质激素受体-α(hGRα-Myc/DDK, +)或Myc/DDK-标记人全长盐皮质激素受体(hMR-Myc/DDK, +)的构建体转染,使用的抗体为Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (上图) 和DYKDDDDK Tag Antibody (与Sigma的Anti-FLAG® M2 Antibody结合相同的抗原表位) #2368 (下图).

Chromatin IP

Chromatin IP

A549 cells were cultured in media with 5% charcoal-stripped FBS for 3 days and then either untreated (left panel) or dexamethasone-treated (100 nM, 1 hr; right panel). Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 cells and 5 µl of Glucocorticoid Receptor (D8H2) XP® Rabbit mAb or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human SLC19A2 Promoter Primers #7681, human MT2A promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.A549培养在含5%活性炭/葡聚糖处理FBS的培养基中,培养3天,之后细胞不处理(左面板)或用地塞米松处理 (100 nM, 1 hr; 右面板)。通过交联染色质进行染色质免疫沉淀,交联染色质来自于4 x 106个细胞和5 µl Glucocorticoid Receptor (D8H2) XP® Rabbit mAb或2 µl Normal Rabbit IgG #2729,使用的试剂盒为SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003.通过real-time PCR 方法,对提取的DNA进行量化,使用的引物为SimpleChIP® Human SLC19A2 Promoter Primers #7681、human MT2A promoter primers和 SimpleChIP® Human α Satellite Repeat Primers #4486. 在每个样品中的免疫沉淀的DNA量表示为相当于加入染色质的总量,相当于一个。

Flow Cytometry

Flow Cytometry

Human whole blood was fixed, lysed, and permeabilized as per the Cell Signaling Technology Flow Alternate Protocol and stained using Glucocorticoid Receptor (D8H2) XP® Rabbit mAb (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. Samples were gated on CD3+ lymphocytes.

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 mutiple 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). 糖皮质激素通过与转录因子的核激素受体超家族成员——糖皮质激素受体(GR)/ NR3C1联合,控制细胞增殖、炎症和代谢(1)。GR是由一些保守的结构元件,包括一个COOH-末端配体结合域(其中还包含受体二聚化和激素依赖型基因转录的关键残基),一个含有核定位信号的相邻的铰链区,一个含DNA结合结构域的中央锌指和一个参与配体无关的基因转录的NH 2 - 末端可变区。在没有激素的情况下,一个明显的GR类群以无活性方式通过其监管伴侣蛋白,如热休克蛋白90、热休克蛋白70和FKBP52,定位到细胞质中。对于激素结合,GR从分子伴侣复合物释放并作为二聚体转移到细胞核,与特定的DNA序列一起被称为糖皮质激素反应元件(GREs),增加或抑制特定的靶基因转录(2)。据证明,GR-介导的转录激活受磷酸化调节(3-5)。虽然GR可以在没有激素的情况下初级磷酸化。建议GR激素依赖型磷酸化可能决定靶启动子的特异性、辅助因子相互作用、受体信号的强度和持续时间、受体的稳定性和受体的亚细胞定位(3)。事实上,GR磷酸化更大程度上是在激素和生化分馏研究存在的情况下,可在细胞核中发现激素治疗表明丝氨酸(211位)-磷酸化GR(3)。因此,丝氨酸(211)磷酸化是体内激活GR的生物标志物。GR信号附加层的复杂性在于通过选择性剪接和使用选择性翻译启动起始位点产生多发亚型的能力,从而增加了功能信号同源和异源二聚体库(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.

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