Active Recombinant Full Length Human STAT3, His-tagged

• Cat.No.: STAT3-29823TH
• Product Overview: Recombinant full length Human STAT3 with His tag, Predicted MWt 89 kDa.

Specification

• Species: Human
• Source: E.coli
• Tag: His
• Description: The protein encoded by this gene is a member of the STAT protein family. In response to cytokines and growth factors, STAT family members are phosphorylated by the receptor associated kinases, and then form homo- or heterodimers that translocate to the cell nucleus where they act as transcription activators. This protein is activated through phosphorylation in response to various cytokines and growth factors including IFNs, EGF, IL5, IL6, HGF, LIF and BMP2. This protein mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. The small GTPase Rac1 has been shown to bind and regulate the activity of this protein. PIAS3 protein is a specific inhibitor of this protein. Three alternatively spliced transcript variants encoding distinct isoforms have been described.
• Conjugation: HIS
• Tissue specificity: Heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.
• Biological activity: 1 unit equals 1 nanogram of purified protein.
• Form: Liquid
• Purity: >95% by SDS-PAGE
• Storage buffer: Preservative: NoneConstituents: 20% Glycerol, 20mM Tris HCl, 100mM Potassium chloride, 1mM DTT, 0.2mM EDTA, pH 7.9
• Storage: Shipped on dry ice. Upon delivery aliquot and store at -80oC. Avoid freeze / thaw cycles.
• Sequence Similarities: Belongs to the transcription factor STAT family.Contains 1 SH2 domain.
• Full Length: Full L.
• Publications:
  An FDA-Approved Antifungal, Ketoconazole, and Its Novel Derivative Suppress tGLI1-Mediated Breast Cancer Brain Metastasis by Inhibiting the DNA-Binding Activity of Brain Metastasis-Promoting Transcription Factor tGLI1 (2021)
  TrkA Interacts with and Phosphorylates STAT3 to Enhance Gene Transcription and Promote Breast Cancer Stem Cells in Triple-Negative and HER2-Enriched Breast Cancers (2021)

Gene Information

• Gene Name: STAT3 signal transducer and activator of transcription 3 (acute-phase response factor) [ Homo sapiens ]
• Official Symbol: STAT3
• Synonyms: STAT3; signal transducer and activator of transcription 3 (acute-phase response factor); signal transducer and activator of transcription 3; APRF
• Gene ID: 6774
• mRNA Refseq: NM_003150
• Protein Refseq: NP_003141
• MIM: 102582
• Uniprot ID: P40763
• Chromosome Location: 17q21
• Pathway: Acute myeloid leukemia, organism-specific biosystem; Acute myeloid leukemia, conserved biosystem; Adipocytokine signaling pathway, organism-specific biosystem; Adipocytokine signaling pathway, conserved biosystem; Adipogenesis, organism-specific biosystem
• Function: CCR5 chemokine receptor binding; DNA binding; calcium ion binding; glucocorticoid receptor binding; ligand-regulated transcription factor activity

Citation

An FDA-Approved Antifungal, Ketoconazole, and Its Novel Derivative Suppress tGLI1-Mediated Breast Cancer Brain Metastasis by Inhibiting the DNA-Binding Activity of Brain Metastasis-Promoting Transcription Factor tGLI1

Journal: Cancers    Data: 2022/8/31

Authors: Daniel Doheny, Sara Manore, Derek Radisky

Article Snippet:Approximately 600 ng of recombinant STAT3 (Creative BioMart, STAT3-29823TH) (Shirley, NY, USA), GLI1 (Creative BioMart, GLI1-312H), or N-tGLI1 protein was mixed with 5X binding buffer (50 mM Tris pH 7.5, 50 mM NaCl, 200 mM KCl, 5 mM MgCl 2 , 10 mM EDTA, 5 mM DTT, 250 μg/mL BSA, 25% glycerol), 50 ng/μL poly dI·dC (Sigma P4929), and 5 pmol 6FAM-labeled dsDNA oligo (Integrated DNA Technologies, Coralville, IA, USA) in a total reaction volume of 20 μL.. The oligos were ordered as the dsDNA from IDT with the sequences /56-FAM/CGAAGA GACCACCCA GGTAGCT and /56-FAM/AGCTACC TGGGTGGTC TCTTCG; the GLI1 consensus binding sequence is underlined.The oligos were ordered as the dsDNA from IDT with the sequences /56-FAM/CGAAGA GACCACCCA GGTAGCT and /56-FAM/AGCTACC TGGGTGGTC TCTTCG; the GLI1 consensus binding sequence is underlined.

KCZ and the novel derivative KCZ-7 inhibit tGLI1 transcriptional activity leading to downregulation of validated tGLI1-mediated stemness genes Nanog and OCT4 . ( a ) Representative Western blots of GLI1 and tGLI1 expression in isogenic SKBRM cell lines following 24 h treatment with vehicle, 1 μM KCZ, or 1 μM KCZ-7. The same membrane was probed to assess the loading control. ( b ) Western blots of recombinant GLI1 and N-tGLI1 (left). A tGLI1-selective Ab was used to detect tGLI1. Binding of recombinant GLI1 and N-tGLI1 to a dsDNA oligonucleotide containing the consensus GLI1/tGLI1-binding site (right). STAT3 was used as a negative control. ( c ) The DNA-binding ability of recombinant N-tGLI1, but not GLI1, is disrupted by KCZ or KCZ-7 treatment. ( d ) Relative binding of GLI1 or tGLI1 to the GLI1-binding sites in SKBRM cells, as determined by chromatin immunoprecipitation; qPCR was performed using primers spanning the GLI1 binding site. ( e , f ) Inhibition of GLI1- and tGLI1-mediated promoter transactivation by KCZ ( e ) and KCZ-7 ( f ). SKBR3 cells were transiently transfected with 8 × 3′GLI1 luciferase reporter and vector, GLI1, or tGLI1 plasmids, then treated with increasing doses of KCZ ( e ) or KCZ-7 ( f ) for 48 h and stimulated with SHH ligand (100 ng/mL) for 4 h. Right: Relative luciferase activity normalized to vehicle treatment. ( g , h ) Selective reduction of tGLI1-mediated stemness genes Nanog ( g ) and OCT4 ( h ) mRNA as assessed by RT-qPCR in isogenic SKBRM cell lines treated with vehicle, 1 μM KCZ, or 1 μM KCZ-7 for 24 h. ( i ) Nanog and OCT4 protein expression following treatment with vehicle, 1 μM KCZ, or 1 μM KCZ-7 in isogenic SKBRM cell lines. The same membrane was probed to assess the loading control. ( j , k ) Overexpression of Nanog ( j ) or OCT4 ( k ) rescues SKBRM-tGLI1 mammospheres from KCZ and KCZ-7 treatment. Scale bars represent 200 μm. N-tGLI1, N-terminal tGLI1; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; two-way ANOVA with post hoc Dunnett’s ( d – f ) or Bonferroni’s ( g , h , j , k ) multiple comparison test was used to calculate p -values. The uncropped blots are shown in page 2 of .