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人 GSK3B 基因ORF全长cDNA克隆(表达载体), C-Flag 标签

  • Human glycogen synthase kinase 3 beta Gene cDNA Clone (full-length ORF Clone), expression ready, C-FLAG-tagged
  • Human glycogen synthase kinase 3 beta natural ORF mammalian expression plasmid, C-Flag tag
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 GSK3B cDNA Clone产品信息
NCBI注册码:NM_001146156.1
参考序列ORF长度:1263bp
cDNA基因描述:Full length Clone DNA of Homo sapiens glycogen synthase kinase 3 beta with C terminal Flag tag.
分子别称:GSK3B
分子种属:Human
载体:pCMV3-C-FLAG
质粒:pCMV3-GSK3B-Flag
限制性酶切位点:KpnI + XbaI (6kb + 1.31kb)
Tag序列:FLAG Tag Sequence: GATTACAAGGATGACGACGATAAG
序列信息:Identical with the Gene Bank Ref. ID sequence.
测序引物:T7(TAATACGACTCACTATAGGG) BGH(TAGAAGGCACAGTCGAGG)
( We provide with GSK3B qPCR primers for gene expression analysis, HP100137 )
启动子:Enhanced CMV mammalian cell promoter
Application:Stable or Transient mammalian expression
抗生素(大肠杆菌):Kanamycin
抗生素(哺乳动物细胞):Hygromycin
运输方式:Each tube contains lyophilized plasmid.
储存方法:The lyophilized plasmid can be stored at room temperature for three months.
 GSK3B 基因质粒图
Human glycogen synthase kinase 3 beta Gene cDNA Clone (full-length ORF Clone), expression ready, C-FLAG-tagged
 GSK3B 基因表达验证图
Human glycogen synthase kinase 3 beta natural ORF mammalian expression plasmid, C-Flag tag
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Caption:
The plasmid was transfected into 293H adherent cells with Sinofection reagent (Cat# STF01). After 48 h, Immunofluorescence staining of cells. Cells were fixed with 4% PFA, permeabilzed with 0.3% Triton X-100 in PBS, blocked with 10% serum, and incubated with Mouse anti-Flag Tag monoclonal antibody (CST#8146S) at 37℃ 1 hour. Then cells were stained with Goat Anti-mouse IgG secondary antibody. The fluorescent signal is detected by fluorescence microscope. Each expression experiment has negative control.
FLAG Tag Info

FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits.

A FLAG-tag can be used in many different assays that require recognition by an antibody. If there is no antibody against the studied protein, adding a FLAG-tag to this protein allows one to follow the protein with an antibody against the FLAG sequence. Examples are cellular localization studies by immunofluorescence or detection by SDS PAGE protein electrophoresis.

The peptide sequence of the FLAG-tag from the N-terminus to the C-terminus is: DYKDDDDK (1012 Da). It can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag or Myc-tag. It can be fused to the C-terminus or the N-terminus of a protein. Some commercially available antibodies (e.g., M1/4E11) recognize the epitope only when it is present at the N-terminus. However, other available antibodies (e.g., M2) are position-insensitive.

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研究背景

GSK3B is a serine-threonine kinase, belonging to the glycogen synthase kinase subfamily. It Contains 1 protein kinase domain, and is expressed in testis, thymus, prostate and ovary and weakly expressed in lung, brain and kidney. GSK3B is involved in energy metabolism, neuronal cell development, and body pattern formation. Polymorphisms in GSK3B gene have been implicated in modifying risk of Parkinson disease, and studies in mice show that overexpression of this gene may be relevant to the pathogenesis of Alzheimer disease. GSK3B participates in the Wnt signaling pathway. It is implicated in the hormonal control of several regulatory proteins including glycogen synthase, MYB and the transcription factor JUN. Phosphorylates JUN at sites proximal to its DNA-binding domain, thereby reducing its affinity for DNA. Phosphorylates MUC1 in breast cancer cells, and decreases the interaction of MUC1 with CTNNB1/beta-catenin. GSK3B also plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex. It prevents the phosphorylation of APC and CLASP2, allowing its association with the cell membrane. In turn, membrane-bound APC allows the localization of MACF1 to the cell membrane, which is required for microtubule capture and stabilization. GSK3B phosphorylates MACF1 and this phosphorylation inhibits the binding of MACF1 to microtubules which is critical for its role in bulge stem cell migration and skin wound repair. It may be required for early embryo development and neuron differentiation.

参考资料
  • Bergmann C, et al. (2011) Inhibition of glycogen synthase kinase 3β induces dermal fibrosis by activation of the canonical Wnt pathway. Ann Rheum Dis. 70(12):2191-8.
  • Ban JO, et al. (2011) Troglitazone, a PPAR agonist, inhibits human prostate cancer cell growth through inactivation of NFκB via suppression of GSK-3β expression. Cancer Biol Ther. 12(4):288-96.
  • Tsukigi M, et al. (2012) Re-expression of miR-199a suppresses renal cancer cell proliferation and survival by targeting GSK-3β. Cancer Lett. 315(2):189-97.
  • Nandan D, et al. (2012) Myeloid cell IL-10 production in response to leishmania involves inactivation of glycogen synthase kinase-3β downstream of phosphatidylinositol-3 kinase. J Immunol. 188(1):367-78.
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